Identification of Single-Base Mismatches in Pneumocystis jirovecii Isolated from Iranian TB positive Patients by CSGE Heteroduplex

Saeideh AmaniGhayoum¹, Masoomeh Shams-Ghahfarokhi^1*, Mehdi Razzaghi-Abyaneh² and Behnam Mohammadi Ghalehbin³

 ¹Department of Mycology, Faculty of Medical Sciences, TarbiatModares University, Tehran 14115-331, Iran.
²Department of Mycology, Pasteur Institute of Iran, Tehran 13164, Iran.
³Department of Medical Parasitology, Ardabil University of Medical Sciences, Ardabil, Iran.

ABSTRACT

Pneumocystis jirovecii is an opportunistic fungus, which causes Pneumocystispneumonia (PJP) in immunocompromised, COPD and TB positive patients with a high rate of colonization, morbidity and mortality. Dihydropteroate synthase (DHPS) gene mutations are well-reported  in PJP. Although sulfa prophylaxis generally is associated with DHPS mutant infection, Multiple molecular techniques applied for detect sulfa resistance single-base mutation.Conformation sensitive gel electrophoresis (CSGE) is a rapid screening method for detection of DNA sequence variation, specifically single-base changes or small insertions and deletions. The current study is investigate on the DHPS sequence single-base dislocation amongstrains isolated from Iranian TB positive co-infected with  PJP  in association to increased levels of serum Lactate Dehydrogenase. Through high serum lactate dehydrogenase (LDH) levels have been associated with established Pneumocystis pneumonia. We investigated the DHPS mismatches infive P.jirovecii isolated of TB infected patients.For genetic identification of Pneumocystis isolates and detection of intraspecific variation, we developed a method for heteroduplex analysis. Our utilizing fragments was the DHPS gene regions, amplified by PCR methodwith specific primers. Serum LDH indicator was analysed for lung acute damages. In our results, at least 4 suspected isolates show more slowly migrating bands containing single heteroduplexes, reveal single-basemislocation in studied sequences.LDH level Peak was higher (p<0.05) in patients with PCP (445± 155 U/L) in comparison patients with chronic TB (310±50 U/L).CSGE is a simple manual method, based on heteroduplex analysis, and compares well in terms of sensitivity with other screening technologies. Manual CSGE remains a low-cost, accessible, and effective approach for mutation screening, which can be carried out with ­minimal specialist equipment.

Keywords: Pneumocystis jirovecii, Dihydropteroate synthase (DHPS), Gene mutations, CSGE, Heteroduplex,Lactate dehydrogenase (LDH).