Infantile Nosocomial Myiasis in Iran
A 5.5-year-old Iranian girl was referred from Gonbad County, Golestan Province, northern Iran to the Shaheed Rajaei Heart Center in Tehran. She was operated on her heart ten days later and then was delivered to Intensive Care Unit (ICU) of the hospital. In this part of the hospital a live parasite was found in her nasal secretions suction and consignment to the department of medical entomology in the School of Public Health, Tehran University of Medical Sciences, Iran. Putative patient died 45 days after admission, unfortunately. Since this infestation manifested more than 40 days after the patient hospitalization, by definition, this may be considered nosocomial. Current morphological characters as well as sequence of a part of cytochrome oxidase subunit one of mitochondorial DNA (mtDNA-COI) was used for identification of the mentioned dipterous larva.\nThe most important dates for this myiasis report were: patient hospitalization, 25 August 2010, open heart operation, 4 September 2010, parasite separation, 5 October 2010, and expiration, 9 October 2010.\nThe key morphological characters including size of the larva, spinous bands, position of posterior spiracles and posterior cavity as mentioned by Zumpt () examined under microscope (Olympus SZX12). The cephalo-pharyngeal skeleton and both anterior and posterior spiracle dissected and mounted on glass slide by Pouri solution. After desiccation the larva was keyed using valid keys (, , , , ). Testing the morphological characteristics revealed that the size of the larva was as long as 5 mm, spinous bands with sclerotized spines, the posterior spiracles located in deep posterior cavity, prothoracic spiracles with 9 openings branches, peritreme present, with 2 distinct slits, spiracular slites pointing toward opening in peritreme, posterior spiracles with the incomplete peritreme, and dorsal arm of cephalo-skeleton longer than ventral arm (–). On the basis of above characters the larvae presumably identical to the second instar larva of W. nuba (Diptera: Sarcophagidae).\nExcept the cephalo-pharyngeal skeleton and spiracle the rest of the larva body was used for DNA extraction using the QIAGEN-DNeasy Blood and Tissue Kit (Germany). The mtDNA-COI gene extending 690 bp of 5′ fragment as applied by Lunt () was amplified using primers including C1-J-2090/C1-N-2735. This region of gene was amplified in 20μl reaction mixtures containing 1μl each of the forward and reverse primers, 1μl the genomic DNA, 1U of Taq DNA polymerase, 1.5 Mm MgCl2 and 250 μm dNTPs. The PCR amplification was performed in two thermal circulations. After initial denaturation at 94 °C for 2 min, the first one performed for 5 cycles of 94 °C for 40 s, 45 °C for 40 s, and 72 °C for 1 min, and the last one repeated for 35 cycles of 94 °C for 40 s, 51 °C for 40 s, and 72 °C for 1 min. Final extension step was continued at 72 °C for 5 min. PCR products were visualized on a 1% agarose gel containing ethidium bromide and using an UV transilluminator. The PCR products were sequenced by Seqlab, a German sequencing company. Sequence of this fragment was deposited in the GenBank database with accession number JF277565.\nThe obtained sequence in this study was compared with the relevant sequences available in public databases using the BLAST program () and then were aligned using ClustalW2 ().\nPhylogenetic relationships between the specimen of this study and some representatives of other related flies causing myiasis including EU418542, AB112857, AB112861, AJ426041, EU418536, DQ345078, AF083657, AB1128 51, AB112854, EU418545, AJ417708, AF295 559, AF104625, EU627697, and EU627705 were inferred with the algorithms implemented in ClustalW2. Sequence of COI region of two member of Muscidae (Musca ventrosa: EU 627697, Neomyia timorensis: EU627705) were used as out groups.\nSequence comparison of the specimen with the available data in Genbank using BLAST revealed maximum 93% identity with Chrysomya albiceps COI region. Other entries of the COI sequences with similarity less than 93% belonged to other dipteran flies of Calliphoridae, Muscidae, Scathophagidae and Drosophilidae families. The phylogenic trees inferred from these data showed that the specimen placed in a completely isolated branch far from other dipteran species particularly from the closely related species of Calliphoridae (). However, among different branches on the tree, Protocalliphora sialia (Calliphoridae) was the most associated fly to W. nuba.