the genus dirofilaria consists of at least 2 species of epidemiologically important human pathogens , dirofilaria immitis ( leidy , 1856 ) and dirofilaria ( nochtiella ) repens ( railliet and henry , 1911 ) .
these worms commonly cause dirofilariasis in dogs , cats , and wild carnivores   and are globally recognized as etiological agents of zoonotic infections in humans , transmitted by vectors / mosquitoes ( mosquito - borne zoonosis ) , including anopheles , culex , and aedes .
d. immitis , that causes dirofilariasis in dogs , cats , and humans , tends to distribute in temperate and tropical regions of the world , while d. repens infections are limited in asia and europe , including vietnam .
however , to our knowledge , no previous report of human infection on the posterior thoracic region due to d. repens has been reported . here
, we report a rare case of subcutaneous posterior thoracic infection caused by d. repens diagnosed by morphology and molecular analysis .
on 14 march 2011 , a 34-year old woman , visited a surgeon at a clinic in a suburban area of hanoi , vietnam with a painful swelling and itchy and palpable nodule on her posterior thoracic wall .
she lived in an area where dogs and cats were outdoors allowed and several times exposed to mosquito bites .
three weeks before , on 26 february 2011 , she suddenly noticed a swollen , tangible , subcutaneous nodule of the similar appearance as the current one at the same site but it has spontaneously disappeared days after . during the physical examination ,
a smooth , tangible , slightly movable , painful nodule of 12 cm in size was touchable under the skin on the posterior part of the thoracic wall , correlative with the vertebrae d11 .
the overlying skin was normal without any overt signs of inflammation and axillary lymph nodes were not enlarged .
routine laboratory test showed red blood cells of 3.910/mm , white blood cells of 6.110/mm ( the percentage of neutrophils , lymphocytes , and eosinophils were 60% , 20.7% , and 11.5% , respectively ) .
no microfilaraemia was detected at this time in her blood samples by microscopy . by local excision under anesthesia ,
after opening the capsule , a thin thread - like worm was noted and captured in 3 fragments which then were fixed in 10% formalin for morphological examinations and a piece was preserved in 70% ethanol for molecular identification . a long , slender , whitish worm ( fig .
1a , b ) excised from the nodule was approximately 10.5 cm in length and about 400 m in its largest width , typical of the description of a female d. repens .
microscopy revealed a thick laminated cuticle with the characteristic longitudinal ridges and cross - striations ( fig .
the anterior end was bluntly rounded and was of greater diameter than the posterior end ( fig .
1a ) . the body cavity contained a female reproductive system , with the bulbous vulva of about 1 mm from the anterior end , and ended in a uterine bifurcation . following the surgical removal , symptoms were quickly resolved , and the patient was recovered without any consequences after a long - time followed up .
total genomic dna was extracted from an ethanol - fixed fragment of the excised worm by qiaamp dna mini kit ( qiagen , hilden , germany ) .
pcr of a portion of the mitochondrial cytochrome c oxidase i ( cox1 ) gene was performed using the primer pairs ( forward dimref , 5  gatgttgcctttgatattggg 3  ; and reverse dimrer , 5  accagcwgtwyacatatgatgacc 3  ) designed based on the alignment of cox1 sequences of both dirofilaria repens and d. immitis available in genbank .
the pcr , of 50 l containing 25 l pcr master mix from promega , 2 l of each primer ( 10 pmol/l ) , 2.5 l dmso , 16.5 l pure water , and 1 l template ( 100 ng/l ) , was carried out in a mj thermal cycler ptc-100 ( mj research , watertown , massachusetts , usa ) with initiation at 94c for 4 min , then 35 cycles including denaturation at 94c for 30 sec , annealing at 55c for 30 sec , extension at 72c for 1 min ; and a final cycle of 7 min at 72c to obtain a pcr product of 643 bp , and a 576 bp portion was used for alignment for taxonomic identification . to support the identification , the presence of 16s rrna sequence of wolbachia sp .
was obtained by pcr with the primer pairs designed hereby in this study , based on 16s ribosomal sequence of wolbachia pipientis available in genbank ( wp16f : 5-ccacactggaactgagatacg-3  and wp16r : 5-tgtagccaccattgtagcac-3  ) .
the parasite recovered from the subcutaneous tissue of our patient was identified as d. repens on the basis of the morphology and molecular methods .
the external longitudinal cuticular ridges differentiate d. repens from d. immitis , the other important human pathogens of the genus dirofilaria .
however , identification of dirofilaria spp . based only on the morphology is difficult ; therefore , molecular methods supported to determine the species .
molecular identification was carried out by using primers of cox1 and of nuclear internal transcribed spacer 2 ( its2 ) , and additionally 16s rrna identification of the endosymbiont w. pipientis commonly co - existing with dirofilaria spp . .
nucleotide cox1 sequence determination of the vietnamese dirofilarial nematode revealed 96% identity to those of the italian / albanian d. repens strains ( genbank no . am749230-am749234 ; dq358814 ; aj271614 ; jf461458 ) ; and 89% to d. immitis ( genbank : am749226 ; am749227 ; am749229 ; aj537512 ; eu159111 ; eu163945 ; kc107805 ; aj271613 ; dq358815 ; fn391553 ) of different geographic origins .
the cox1 amino acid sequence showed 99 - 100% similarity to all of the d. repens samples , while only 96% to d. immitis .
high intraspecific variability was observed in d. repens 
 cox1 sequences , while those of d. immitis species samples were identical ( 100% identity to each other ) , regardless of geographical distribution .
phylogenetic tree inferred from the 576 bp mitochondrial cox1 sequence of 20 strains including d. repens collected from the subcutaneous posterior thoracic nodule in this study , and 8 other d. repens , 10 d. immitis , and a nematode species o. lupi ( as an out - group ) indicated that the vietnamese filarial sample clustered with cox1 sequences of d. repens , in a group , that was completely separated from the d. immitis and from o. lupi out - group sequences ( fig .
the genetic distances among the newly identified d. repens of vietnamese patient and reference strains were calculated based on the kimura 2-parameter model using mega5.2 software   ( table 1 ) .
the genetic distances among the dirofilaria strains within each of d. repens or d. immitis , respectively , were very low , ranging from 0.000 to 0.039 for d. repens ( table 1 , no . 1 - 9 ) and no variation ( 0.000 ) for d. immitis ( table 1 , no .
there was relatively high taxonomic genetic distance , ranging from 0.103 to 0.117 between d. repens and d. immitis ( table 1 , no . 1 - 9
10 - 19 ) ; and 0.109 to 0.125 between dirofilaria spp . and a nematode o. lupi , used for an out - group ( table 1 , no . 20 ) .
by amplification with the primer pairs based on 16s ribosome , a sequence of 918 nucleotides was obtained and compared with those of the previously known w. pipientis 16s rrna sequences , i.e. , one amplified from a d. repens ( italian strain , genbank : aj276500 ) and several from d. immitis strains ( z49261 , af088187 , and af487892 ) , available in genbank .
the comparative sequence analysis revealed 96 - 97% identity among the endosymbionts and accurately confirmed the presence of the endosymbiont w. pipientis in the vietnamese d. repens .
the species identification of this vietnamese d. repens , thus , was completely accurate using both the mitochondrial and nuclear nucleotide analysis and distance - based / phylogenetic approaches . to date , of 3,192 human dirofilariasis cases reported worldwide , 372 pulmonary and 1,410 subcutaneous / ocular infections were documented ; and due to aberrant migration of the microfilariae , dirofilaria spp .
lesions caused by d. immitis are focused mainly in the heart ( dogs and cats ) and in the lungs and skin ( in humans , presented as pulmonary and subcutaneous forms ) , whilst d. repens tends to localize in multiple organs , mainly in the subconjunctiva , lungs and coronary artery [ 11 - 13 ] , soft tissues as the breast , penis , spermatic cord , epididymis , and scrotum [ 14 - 17 ] , brain , liver , lymph nodes , muscles , and anterolaterial neck [ 5,18 - 21 ] , and rare places , i.e. , causing intravitreous infection in humans . to our knowledge , this is the first case of human infection on subcutaneous posterior thoracic region due to d. repens reported . in humans , normally a single subcutaneous nodule and lesions have been developed during weeks and described as little granulomatous inflammatory lesions . in the case
reported here , the time and the means of transmission by which the patient caught d. repens infection is unclear , probably by mosquitoes bites ( infected by larvae from the outdoor dogs / cats wandering in the suburb ) .
unfortunately , no surveys of microfilariae in dogs / cats have been conducted yet . to date , 12 dirofilaria infections of the conjunctiva ( eye subcutaneous and subconjuctiva ) have been detected in vietnam .
the case of posterior thoracic localization hereby presented is a rare record and all of the vietnamese infections documented are autochthonous , caused by solitary d. repens . in our case ,
molecular classification of the causal agent confirmed it to be d. repens using complex approaches , including morphological , historic , mitochondrial cox1 , nuclear its-2 , distance - based / phylogenetic analysis , and supported by the endobiosymbiont , w. pipientis , 16s - rrna based identification .
regarding high risk of human dirofilariasis ( imported dirofilaria is not exclusive ) in vietnam , detailed national surveys involving pathological , epidemiological and etiological information on dirofilaria species , animal / host reservoirs , vectors / mosquitoes , provincial distribution and improvement of routine laboratory diagnostic approaches , including molecular identification / discrimination of zoonotic dirofilaria are needed for early control of the emerging dirofilarial infections .
 the emergence of dirofilarial infections in asia including vietnam is a clinically significant threat to the community . we here report a rare case of subcutaneous dirofilaria repens infection on the posterior thoracic wall in a young woman presenting a painful , itchy , and palpable nodule . 
 the adult worm was identified by mitochondrial cox1 and nuclear its-2 sequence determination . 
 the diagnosis was additionally confirmed by 16s rrna sequencing of the endosymbiont wolbachia pipientis commonly co - existing with d. repens . 
 this is a rare case of subcutaneous human infection on the posterior thoracic region caused by d. repens . 
INTRODUCTION
CASE RECORD
DISCUSSION