Biomed Environ Sci, 2021; 34(3): 247-249 247 Letter to the Editor Investigating the Aggregation of Imported Cutaneous Leishmaniasis in Henan, Central China* ZHAO Yu Ling1,#, LI Su Hua1, ZHU Xin2, ZHOU Rui Min1, YANG Cheng Yun1, LIU Ying1, NIU Yuan Na1, LU De Ling1, ZHANG Hong Wei1, and ZHAO Dong Yang1 Leishmania is a parasitic disease caused due to lacked the knowledge of its clinical manifestations, infection with Leishmaniasis, which is prevalent in 88 which often led to misdiagnosis. countries across the globe[1]. Clinically, The Republic of Uzbekistan is a landlocked Leishmaniasisis is divided into 3 types: visceral country located in Central Asia. In October 2017, an leishmaniasis (VL), mucosal cutaneous leishmaniasis employee (Bai XX) who returned from the (MCL), and cutaneous leishmaniasis (CL)[2]. VL, which construction site of Uzbekistan was diagnosed with is known as kala-azar, is caused by Leishmania CL in Zhengzhou Sixth People’s Hospital. This is the donovani specie (L. donovani); and the infected first case of CL reported in Zhengzhou City in the patient's skin often shows dark pigmentation and past 50 years. In July 2017, Bai XX’s arms, back, fever, hence the name kalaazar. In China, VL is dorsum of hands, and other parts showed swelling, considered as the most serious disease and is ulceration, and pigmentation spots on the skin. In prevalent in Xinjiang, Inner Mongolia, Gansu, Karakuri, Uzbekistan, patients were diagnosed with Sichuan, Shaanxi, and Shanxi provinces[3]. CL is rarely CL in a local hospital. After hospitalization and reported in China, as there were only 3 reported treatment, he was discharged from the hospital on cases of CL in China since 2010, while the imported October 16, 2017. Later, some other employees CL cases were caused by L. major[4,5]. showed similar symptoms. By January 2018, 9 CL is mainly caused by Leishmania tropicalis or people had skin damage. After returning to China, Leishmania gigantica, which is related to the bite of Bai XX visited the Zhengzhou Sixth People’s Hospital infected female phlebotomine sandflies. CL is an on October 30 due to skin swelling, erosion, and epidemic disease, with India, North Africa, and ulcers; and was diagnosed with CL. In addition, in Mediterranean coastal countries representing the December 2017, Zhang XX had scabby which exuded endemic regions[6,7]. In China, CL is only found in under the scab on the left clavicle. Yu XX had brown Shawan and Karamay in Jungar basin, as well as in inflammatory spots on the popliteal fossa of his left Northern Tianshan Mountains, Xinjiang. Since 1949, a leg (Figure 1). The other 6 people had skin damage large-scale diagnosis and control of important parasitic but were not seriously infected. After treatment in diseases that endanger human health have been foreign countries, the skin was not completely cured. carried out in China. However, with the increase in Henan Center for Disease Control (CDC) and number of overseas workers, the number of imported Prevention paid close attention to these imported CL cases has increased sharply and the sources of cases and handled the epidemic situation by infection are mainly countries with relatively high pathogen diagnosis, treatment of patients, and prevalence of CL, such as North Africa, as well as vector investigation. According to the information Middle East and Central Asia[12]. After 1984, except in reported by the network of epidemics of infectious Zhengzhou in 2011, a case with unknown source of diseases, the infection was traced back to the time infection was found and there were no local cases of of exposure. From December 2017 to January 2018, leishmania infection in Henan Province. In the past 50 we conducted screening and etiological diagnosis of years, the incidence of CL is low and many clinicians leishmaniasis for 180 migrant workers of the same doi: 10.3967/bes2021.032 *This study was funded by Grants from the department of Science and Technology of Foundation of Henan province, China [No.162102310035] and Henan provincialhealth and family planning science and technology innovative talents project [51282]. 1. Henan Key Laboratory of Infectious Disease Microbiology, Henan Center for Disease Control and Prevention, Zhengzhou 450016, Henan, China; 2. Luoyang Center for Disease Control and Prevention, Luoyang 471023, Henan, China 248 Biomed Environ Sci, 2021; 34(3): 247-249 construction project. The basic information of these obtained was stored at 20 °C for until further use. workers were collected. Primers K13A-K13B and L5.8S-LITSR were used for Blood test, skin lesions examination, and rK39 kDNA and ITS1 amplification in ribosomal DNA, test were performed for 180 workers. For Leish rK39 respectively (Table 1)[8,9]. test, in brief, 20 μL of serum was added to Leish rK39 The polymerase chain reaction (PCR) system dipstick paper card and then 2–3 drops of chase (50 μL) is as follows: 2× PCR Master Mix 25 μL; buffer solution was added to a test tube followed by forward and reverse primers (1.5 μL each); DNA addition of the dipstick into the test tube containing template of 5 μL (50 ng/μL); and ddH2O the chase buffer and readings were taken after supplemented to 50 μL. The reaction conditions 10 minutes. Among the 180 subjects, 18 had were: 94 °C for 3 min; 94 °C for 30 s; 58 °C for 30 s abnormal findings; whereas 9 had varying degrees of (52 °C for L5.8S-LITSR); 72 °C for 1 min (45 s for skin damage, including 3 with severe skin lesions. L5.8S-LITSR); (35 cycles L for 5.8S-LITSR); and 72 °C Three out of the 9 cases were positive on rK39 for 5 min. 5 μL amplification products were dipstick test. Five cases had hepatomegaly and subjected to 2% agarose gel electrophoresis. In the splenomegaly, and one case had superficial lymph results, there were about 120 BP and 350 BP node enlargement. fragments that were amplified out, which are The tissue samples of typical skin lesions were consistent with those reported by Doudi M and collected for smear and culture. 75% alcohol was Hejazi SH, et al.[10] (Figure 3). The amplified products used to disinfect the skin lesions and marginal skin of of K13A-K13B and L5.8S-LITSR were sequenced by the patients and a sterile scalpel was used to cut Shanghai Biotechnology Co. Ltd and the sequences open the skin lesion and marginal skin of the were aligned using NCBI database. The two patients. A part of the skin lesion tissue was directly amplification products were 90% and 98% smeared and stained with Giemsa and no homologous to the corresponding sequences of L. amastigotes was observed under an oil microscope. major, respectively. The skin tissues specimens were collected and Luoyang Center for Disease Control and cultured in a sterile NNN medium (Novy-Macneal- Prevention organized the surveillance and Nicolle culture medium) at 25 °C. A small amount of investigated sandflies in its jurisdiction. From April to medium was taken every 2–3 days and Giemsa July, sandflies were collected from various districts staining was performed to observe the presence of and counties and the parasites species were promastigotes and amastigotes under an oil identified as Phlebotomus chinensis using molecular microscope (Figure 2). assays in parasitological positive specimens. Nucleic acid was extracted from positive culture At present, there are four known species of with Qiagen nucleic acid extraction kit according to sandflies that transmit leishmaniasis in China: P. the manufacturer’s instruction. The nucleic acid 3 1 4 2 Figure 2. Leishmania amastigotes and promastigotes in NNN culture solution (Giemsa Figure 1. Skin lesions. The Forearm, Chest, and staining, × 1,000). 1-Flower shape promastigotes; Hand from left to right. 2-Promastigote; 3 and 4-Amastigotes. Table 1. Primer sequences of PCR Gene name Kinetoplast DNA Internal transcribed spacer of rDNA Forward primer K13A: 5′ -GTGGGGGAGGGGCGTTCT-3′ LITSR: 5′-CTGGATCATTTTCCGATG-3′ Reverse primer K13B: 5′-ATTTTACACCAACCCCCAGTT-3′ L5.8S: 5′ -TGATACCACTTATCGCACTT-3′ Product length(bp)120 120 350 Aggregation of imported cutaneous leishmaniasis in Henan, central China 249 chinensis, P. longiductus, P. Wui, and P. alexandri. leishmaniasis, the imported leishmaniasis has Since 1970, no Phlebotomushas been found in the become a potential threat to people's health, with plain areas of Henan province; however, there are the aggravation of population mobility in China. some Phlebotomus in the mountain and hilly areas. Therefore, the relevant departments must attach The remnants of sandflies are mainly Phlebotomus great importance to it. The possibility of CL should chinensis and scaly-beak Sergentomyia. be suspected in migrant workers with skin injury According to the monitoring results of sandflies who return from the endemic regions of in Linzhou City, P. chinensis, Phlebotomus, and scaly- leishmaniasis. beak Sergentomyia were the main species in Henan. Acknowledgement Institute of Parasitic Diseases of Luoyang CDC conducted a sandfly surveillance from the Chinese Center for Disease Control and April to July 2018 in Luoning county and other Prevention, Luoyang Entry-Exit Inspection and jurisdictions and reported that all the sandflies were Quarantine Bureau, Luoyang Health and Family P. chinensis. However, the main vectors of CL are Planning Commission and Luoyang Centerfor Disease Phlebotomus papatasi and Phlebotomus sergenti. Control and Prevention. There is low of risk cutaneous leishmaniasis #Correspondence should be addressed to ZHAO Yu transmission in our province, due to the difference Ling, associate senior doctor. Tel: 86-371-68089136, and blocking of the transmission link of the media. E-mail: [email protected] Dermatologically, this disease is diagnosed as Biographical note of the first author: ZHAO Yu Ling, biting dermatosis. As Leishmania propagates into the female, born in 1968, Master of Pathogeny Biology, lesion tissues, the absence of solustibosan causes majoring in microbiology and molecular epidemiology.