Hussein Eman M, Al-Mohammed Hamdan I, Hussein Abdalla M
Parasitology Department, Suez Canal University, P.O. Box 41152, Ismailia, Egypt.
Parasitol Res. 2009 Oct;105(4):1053-60. doi: 10.1007/s00436-009-1515-9. Epub 2009 Jun 19.
Dientamoeba fragilis is a parasite that has been recognized as a causative agent of gastrointestinal symptoms. The search for genetic variation in D. fragilis based on the small-subunit (SSU) rRNA gene using restriction fragment length polymorphism was found not useful for molecular epidemiology. In this study, genetic variability of different clinical isolates of D. fragilis was explored by high-resolution melting curve (HRM) following polymerase chain reaction (PCR) in a one-step closed-tube method. Thirty fecal samples from irritable bowel syndrome (IBS) patients having D. fragilis trophozoites and negative for other organisms were involved in this study. According to the type of diarrhea, eight patients had acute, 14 patients had chronic intermittent, and eight patients had diarrhea alternating with constipation. HRM proved that four profiles (subtypes) were present as detecting by scanning mutation. One of these profiles (profile 1) was predominant (50%). Profile 2 was present on 20%. Profiles 3 and 4 were present on 16.7% and 13.4%, respectively. No mixed profiles were detected among the samples. The melting curves characterized by T(m)1=77.17+/-0.29 degrees C in profile 1, T(m)1=77.37+/-1.45 degrees C in profile 2, T(m)1=74.24+/-0.08 degrees C and T(m)2=79.64+/-0.09 degrees C in profile 3, and T(m)1=75.51 +/- 0.09 degrees C and T(m)=79.42 +/- 0.09 degrees C in profile 4. The relation between these profiles and types of diarrhea proved that the majority of patients having profile 1 (73.4%) and profile 4 (75%) had chronic intermittent diarrhea. All of the patients having profile 2 had acute diarrhea while all of the patients having profile 3 had diarrhea alternating with constipation. Although profile 1 was detected among all types of diarrhea, it was corresponding to 11/14 of patients with chronic intermittent diarrhea. All the differences were clinically and statistically significant. In conclusion, HRM following PCR was proved as a wide variation on D. fragilis genotypes that could be related to the characters of diarrhea among IBS patients. As the differences in HRM reflect different sequences of SSU RNA gene, thus, another study for identifying the sequences of these isolates (profiles) will be done and published later.
脆弱双核阿米巴是一种已被确认为胃肠道症状病原体的寄生虫。基于小亚基（SSU）rRNA基因，利用限制性片段长度多态性来寻找脆弱双核阿米巴的基因变异，结果发现其对分子流行病学并无用处。在本研究中，采用一步闭管法，通过聚合酶链反应（PCR）后的高分辨率熔解曲线（HRM），对不同临床分离株的脆弱双核阿米巴的基因变异性进行了探索。本研究纳入了30份来自肠易激综合征（IBS）患者的粪便样本，这些样本中含有脆弱双核阿米巴滋养体，且其他微生物检测为阴性。根据腹泻类型，8例患者为急性腹泻，14例患者为慢性间歇性腹泻，8例患者为腹泻与便秘交替。HRM证明，通过扫描突变检测到存在四种熔解曲线图谱（亚型）。其中一种图谱（图谱1）占主导（50%）。图谱2占20%。图谱3和图谱4分别占16.7%和13.4%。样本中未检测到混合图谱。图谱1的熔解曲线特征为T(m)1 = 77.17±0.29℃，图谱2为T(m)1 = 77.37±1.45℃，图谱3为T(m)1 = 74.24±0.08℃和T(m)2 = 79.64±0.09℃，图谱4为T(m)1 = 75.51±0.09℃和T(m)=79.42±0.09℃。这些图谱与腹泻类型之间的关系证明，大多数具有图谱1（73.4%）和图谱4（75%）的患者患有慢性间歇性腹泻。所有具有图谱2的患者均为急性腹泻，而所有具有图谱3的患者均为腹泻与便秘交替。虽然在所有腹泻类型中均检测到图谱1，但它对应于11/14的慢性间歇性腹泻患者。所有差异在临床和统计学上均具有显著性。总之，PCR后的HRM被证明在脆弱双核阿米巴基因型上存在广泛变异，这可能与IBS患者的腹泻特征有关。由于HRM的差异反映了SSU RNA基因的不同序列，因此，后续将开展另一项研究来鉴定这些分离株（图谱）的序列并发表。
