The patient, female, 68 years old, lived in the suburbs of Zhengzhou city, Henan Province, in central China (34°38′ N, 113°11′ E). In January 2019, the woman was admitted to the First Affiliated Hospital of Zhengzhou University complaining of a bitter taste in her mouth and upper abdominal discomfort for more than 6 months. The patient had a history of hypertension and coronary sclerosis. Physical examination showed no obvious symptoms or signs, and her vital signs were stable. Initial laboratory studies showed that her levels of red blood cells, hemoglobin, hematocrit, mean corpuscular hemoglobin, and iron were low, whereas platelets and the blood sedimentation rate were elevated, suggesting iron deficiency anemia. The patient complained that she often worked barefoot in the fields. Gastroduodenoscopy analysis showed several active nematodes in the duodenal bulb (Additional file 1: Figure S1). The worms were collected by endoscopic forceps and identified as N. americanus according to morphological characteristics. More specifically, (1) N. americanus has a slightly rounded buccal cavity provided with a pair of ventral semilunar cutting plates and a pair of smaller dorsal cutting plates (Fig. 1a); (2) a pair of conspicuous neck papillae were present on the ventrolateral margin (Fig. 1b); (3) there were transverse cuticular striations on the body surface (Fig. 1c); (4) the posterior end of the hookworm was rounded and exhibited a copulatory bursa with several papillae on its ventral base (Fig. 1d); and (5) the distal end of the copulatory bursa is divided into two branches, and each branch is further divided into three small rays (Additional file 2: Figure S2). In addition, the cox1 sequences of collected hookworms in this study were identified. All worms generated the identical sequence. The comparison results indicated that the cox1 sequence of hookworms here had a 99.83% similarity with that of the published N. americanus from Yunnan Province in China. For the treatment of ancylostomiasis, the patient was given oral albendazole tablets (GSK Pharmaceuticals Inc., Tianjin, China) 200 mg three times a day for 1 month. During this period, iron and recombinant human erythropoietin were continuously supplemented. The stool occult blood test after treatment showed negative results, and no hookworm eggs were found in the Kato-Katz test. The concentration of serum hemoglobin reached a normal value 2 months after the therapy.
Henan Province has an area of approximately 167,000 km2, with a population of approximately 100 million people. A total of 25,948 cases of hookworm infection in Henan Province were reported from 1949 to 2020, including 25,631 cases (98.78%) from epidemiological investigations and 317 patients (1.22%) diagnosed at hospitals (Fig. 1e, f and Additional file 3: Table S1). Among all reported ancylostomiasis, 2596 cases were caused by N. americanus, and 11,869 cases were caused by A. duodenale. Only one case was reported by simultaneous infection. Most of the reported cases were distributed in southern areas of Henan Province, such as Nanyang and Xinyang. However, no cases have ever been reported in four cities: Hebi, Xinxiang, Sanmenxia, and Jiyuan. According to an analysis by 5-year phases, hospital-diagnosed cases displayed an increasing trend each year from 1949 to 2015 (Additional file 4: Figure S3), but have declined dramatically in recent years (2016–2020), with only four cases reported. For epidemiological cases, from 1949 to 1970, few hookworm infection cases were reported (271 cases). During 1986–1990, the reported ancylostomiasis cases increased dramatically and reached 19,466 cases. However, the number of cases began to decrease year by year after 1990; in particular, in the past 5 years, only 96 epidemiological cases have been reported. Since the first national survey on the distribution of human parasites was carried out in 1990 in China, many provinces including Henan have carried out comprehensive measures for the prevention and control of parasitic diseases in rural areas, focusing on deworming treatment, combined with health education and fecal management [22, 23]. Therefore, the number of cases of ancylostomiasis began to decline significantly after 1990. Generally, although ancylostomiasis cases have dropped substantially in recent years, hookworm infection is still a public health problem that negatively influences health and hinders socioeconomic development in underdeveloped areas and remote rural areas of Henan Province. Positive measures for preventing and controlling ancylostomiasis such as improving the level of knowledge about people’s health and alleviating health poverty are required by both health services and individuals.
In recent years, the genotyping of hookworms based on suitable molecular markers has played an important role in the modern diagnosis and epidemiological investigation of hookworms, and the mt genome can provide more potential markers for molecular identification. In this study, a total of 1,024,362 sequence reads were mapped to the reference mitochondrial genome of N. americanus [10]. The mt genome of the N. americanus collected here was fully annotated and encoded 12 PCGs (cox1-3, nad1-6, nad4L, atp6, and cytb), two ribosomal RNA (rrn) genes, and 22 transfer RNA genes, but lacked an atp8 gene (Fig. 2a). All 36 genes were encoded in identical order and direction, in accordance with other sequenced mt genomes of hookworms [11, 12, 24]. Among the 12 PCGs, the most common initiation codon was ATT (for eight genes), followed by TTG (three genes). Eight PCGs were predicted to have a TAA or TAG termination codon. The remaining protein genes were inferred to end with an abbreviated stop codon, such as T or TA. Incomplete termination codons are common in the mitochondrial genomes of parasitic helminths [24]. Both rrnS and rrnL were 699 bp and 957 bp in length, and located in the positions between tRNA-Glu and tRNA-Ser and between tRNA-His and nad3, respectively. The lengths of the 22 tRNA genes ranged from 52 (tRNA-Ser) to 62 (tRNA-Lys) nucleotides (nt). Among the N. americanus isolates (NC_003416, AJ556134 and this study), the full-length mt genomes were identical in 93.4% nucleotides with the alignment of 13,623 bp in length. Comparing the PCGs, N. americanus isolates displayed high levels of sequence conservation ranging from 91.3% of identical nucleotides in nad1 to 95.4% in cox3. For cox1, the nucleotide identity reached 97.5%. The amino acid sequence similarities ranged from 92.1% (nad1) to 98.5% (atp6). The results of the sequence divergence of PCGs suggest that nad1 shows high sequence variability among isolates, indicating that this gene is suitable as a genetic marker for population genetics studies of N. americanus of different geographical origins. The mt large and small subunit rDNAs (rrnL, rrnS) shared 94.1% and 96.3% identical nucleotides, respectively.
The likelihood models identified by the jModelTest (AIC) suggested that the GTR + G + I model was most suitable for included data partitions. Basal identical tree topologies were generated through phylogenetic inference based on both ML and BI methods. The phylogenetic pattern is demonstrated in Fig. 2b. Hookworm isolates collected here were grouped into two obvious clades: one clade included species of A. caninum, A. tubaeforme, A. duodenale, and A. ceylanicum corresponding to the subfamily of Ancylostomatinae; the other clade contained species of N. americanus, Bunostomum phlebotomum, and B. trigonocephalum corresponding to the subfamily of Bunostominae. Within the Bunostominae, three N. americanus isolates formed a single group and as a sister group with the Bunostomum species, consistent with a recent molecular study [24]. Among N. americanus isolates, isolates from China were more closely related to each other than to that from Togo, indicating that long-distance geographical isolation might accelerate the genetic differences among N. americanus isolates.