Saeideh AmaniGhayoum1, Masoomeh Shams-Ghahfarokhi1*, 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.

KeywordsPneumocystis jirovecii, Dihydropteroate synthase (DHPS), Gene mutations, CSGE, Heteroduplex,Lactate dehydrogenase (LDH).

INTRODUCTION

Pneumocystis jiroveciipneumonia (PJP) formerly known as Pneumocystis cariniipneumonia (PCP) is alife-threatening opportunistic fungal infection of the lungs with a significant morbidity and mortality among immunocompromised patients 1. Pneumocystis often cause of pneumonia in immunocompromised hosts such as hematologic malignancies, organ transplants, immunosuppressive drugs users, pulmonary tuberculosis (TB), COPD andpatients infected with human immunodeficiencyvirus (HIV)2.Tuberculosis makes a special immunebackground with a low supplementary oxygen pressure due to Pneumocystisexpedited colonization3.The emergence of drug resistance is predicted because of the extreme usage of TMP-SMX for prophylaxis against PJP and wide-spectrum antibiotics in respiratory disorders 4. In Pneumocystis sulfa drug resistance gene, singlebase mismatches producetreatment challenges in mutant strains.Finding a proper method todetection of complex gene’s single-base mutations remains a technicalchallenge, and there has been a continuing search for rapid andefficient methods for developed investigations. Although there is several molecular techniques for diversity and mismatch detection, but few was utilize in Pneumocystosis infection.   Conformational sensitive gel electrophoresis (CSGE) is an operational method develope for scanning PCR products for the presence of single-base and larger base transpositions in DNA. The assay was based on the assumption that mildly denaturing solvents in an appropriate buffer can accentuate the conformational changes produced by single-base mismatches in double-stranded DNA and thereby increase the differential migration in electrophoretic gels of heteroduplexes and homoduplexes5. Here the sensitivity of assays by CSGE was improved by limiting the maximal size of the PCR products to 450 bp and making several changes in the conditions for PAGE. With the improved conditions, CSGE detect identified single-base changes in a large series of PCR products that contain multiple exons with highly repetitive and GC-rich sequences and provide a rapid detection of single-base differences in double-stranded PCR products andDNA fragments 6. This analytical method has been used in medical genetics and to clarify the evolution of organisms including viruses, bacteria and human genetic disorders 7. Furthermore we have noted a disconcerting tendencyfor physicians to rely onUsefulness of lactate dehydrogenase and it’s Iso enzymes as serum indicators of chronic lung damage or inflammation. Lactate dehydrogenase (LDH) is a cytoplasmatic enzyme present in essentially all major organ systems. Increased serum/BAL lactate dehydrogenase (LDH) activity is suggesting induced disturbances of cellular integrity that support the presumptive diagnosis of pathological conditions of Pneumocystis pneumonia (PJP) and other inflammations8-9. It may also be increased in other variety of extrapulmonary and granulomatous fungal infections10. We aimed to determine the frequency of infection; the salient clinical, laboratory; and the probable single-base mutations of isolated Pneumocystis inpatients who have this pulmonary co-infection, then find a significant relationship between co-infection morbidity and LDH activity. The general attempt of the present study was to apply heteroduplex analysis for not only routine identification of Pneumocystis jirovecii mutant and drug resistant isolate, but also to modify treatment line and sensitive detection of genetic diversity within species.

MATERIALS AND METHODS

Samples and patients
We study on 5 isolated strain of opportunistic fungi Pneumocystis jirovecii DNA sequences obtain from Tuberculosis co-infected patients in a former retrospective investigation.

Identification of P. jiroveciiand Mycobacterium tuberculosis
Direct microscopic examination of bronchoalveolar lavage (BAL) revealed AFB and culture of the same yielded Mycobacterium tuberculosis. Also, acid fast bacillus was shown by Zeil-Neelsen staining technique and Bronchoalveolar lavage specimens were prepared using cytocentrifugation. Duplicate smears of respiratory specimens submitted for P. jiroveciiexamination and examined for the presence of P. jiroveciicystswith the Giemsa staining technique.

DNA extraction and PCR amplification ofPneumocystis jiroveciidihydropteroate synthase gene
PCR products were synthesized by amplification of DHPS gene according to therecent parallel research11-12. Genomic DNA from P. jiroveciiwas extracted using kit manufactured by Qiagen (QIAampDNA MiniKit; Qiagen, Germany) according to the manufacturer’s instructions.DHPSloci of the P. jirovecii genome were amplified by HUM: 5′-GCGCCTACACATATTATGGCCATTTTAAATC-3′ and DHPS-4: 5′-GGAACTTTCAACTTGGCAACCAC-3’for first PCR and a second specificprimerpairs: Cprim 5’CCCCCACTTATACA-3’andDprim:5′-GGGGGTGTTCATTCA-3’for aninternal amplification 269 bp fragment.

Scanning of the PCR products by CSGE technique
In order to heteroduplex analysis by CSGE, all PCR products(269 bp fragments) were electrophoresedin a 1-mm thick gel with 37-well comb (FMC) prepared with 10 or 15% polyacrylamide, 99:1 ratio of acrylamide (Intermountain Scientific, Kaysville, UT) to 1,4-bis(acryloyl) piperazine (Fluka), 10% ethylene glycol (Sigma), 15%formamide (GIBCO), 0.1% ammonium persulfate (U.S. Biochemicals), and 0.07% N,N,N9,N9-tetramethylethylenediamine (Sigma) in 0.53 TTE buffer. It was important not toautoclave the TTE buffer to obtain optimal separation of heteroduplexes and homoduplexes. Before electrophoresis, EDTA was added to each PCR product in a final concentration of 10 mM. Twenty microliters of each sample was heated to 98°C for 5 min followed by incubation at 65°C for 1 hour to generate heteroduplexes. The optimal polymerization time was about 1 hour. Four microliters of PCR products containing heteroduplexes were mixed with 3 ml ofstock loading buffer (stock solution of 30% glycerol, 0.25% bromphenol blue,0.25% xylene cyanol). Samples were separated by electrophoresis on a standard DNA sequencing gel apparatus with 37.5× 45cm glass plates using 0.53 TTE as the electrode buffer.

Typically, a comb for 37 lanes was used, and up to five PCR products of different sizes were mixed and loaded in each lane. The gel was pre-electrophoresed for 15 minutes, and the samples were separated at room temperature using power as a limitingfactor during the run with 40 W and 6 hours for 10% gels, or 40 W and 8.5 hours for 15% gels. After electrophoresis, the gel was stained on the glass plate in 1 mg/ml of ethidium bromide for 10 min followed by destaining in water5. The relevant section of the gel was cut, transferred to a piece of blotting paper, and then released from the paper onto the surface of a transilluminator by wetting with water. The gel was photographed with either a Polaroid camera or high-quality charge-coupled-device camera for gel documentation (Fotodyne, New Berlin, WI).

Lactate dehydrogenase enzyme assay
In addition, another diagnostic factor was measured as Serum/BAL LDH levelsat the international unit scale (IU) with use of LDH diagnostic kit (PISHTAZTEB), colorimetric assay (BT 3000 system) . In this methodlactate is used as a substrate and NAD as coenzyme. so is the “optimised standard method” according to the recommendations of the German Society for Clinical Chemistry (DGKC). For measurement procedure collected 200 BAL using standard sampling tubes heparinised 125 μland adding 1000 μl of R1 reagent ( tris buffer pH 7.5 50 mmol/l , pyruvate 0.6 mmol/l , preservative),mix, incubate for 1 minute at 37°C temperature, then add: 250 μl of R2 reagent (NADH 0.18 mmol/l , preservative) mix, incubate for 1 minute and read initial absorbance start stopwatch simultaneously, test reaction show in below:

Pyruvate + NADH + H+ ← LDH → Lactate + NAD+

Lactate dehydrogenase catalysis the conversion of pyruvate to lactate; NADH is oxidized to NAD in the process. The rate of decrease in NADH is directly proportional to the LDH activity and is determined photometrically by UV-assay, wavelength: 340 nm, temperature: +37°C, Cuvette: 1 cm, light path andMulticalibrator XL ref: PT-cal, according to a standardized method. Standard reference values for both men and women were 120-240 lU/L 9-10.

Table 1.  Lactate dehydrogenase enzyme alteration between TB positive, negative and co-infected groups

Patient code PJP AFB* LDH(IU/L)
Co-Infected Group 36B + + 443
76B + + 590
30B + + 601
84A + + 476
126A + + 418
TB positive Group 10A + 295
17A + 299
18B + 304
53A + 296
95A + 265
Negative control 31A 238
22B 182
15A 215
152A 135
88B 159

AFB* Test;  Acid Fast Bacillus Test followed by Ziehl-Neelsenstaining technique

RESULTS

Identification of heteroduplex bands of dihydropteroate synthase gene by CSGE
To detect possiblesingle-base mismatches in nucleoside site165 and 171, the conformational electrophoresis analysis was performed on five PCR products that contained of previously identified as a P.jirovecii amplified DHPS gene (Fig.1). Our studied fragments (269 bp) were less than 400 bp so have optimum condition for correct migration.The homoduplexesin all negative control lanes and positive control sequence migrated, but at least 4 suspected isolates show more slowly migrating bandscontaining singleheteroduplexes.Our findings propoundthe geneticalmismatche and diversity in dihydropteroate sequence mutative sites in clinical isolate 1,2,4,5 and confirm diversity in RFLP results of former study in the event that submitted double mutation in codon 55 Thr/57 Pro.

Fig. 1. PCR products of Pneumocystis jirovecii DHPS gene (269bp fragments), used for mutation detection by CSGE technique

Despite the negative report for the third isolation, no heteroduplex production and single-base Displacementwere seen on PAGE even after recheckand doubling the incubation time in the EDTAbuffer (Fig. 2).

Fig. 2. Differential migration of DNA heteroduplexes and homoduplexes. Samples were PCR products (DHPS gene) of five Pneumocystis jiroveciistrains obtained from TB positive patients. Templates contain different base in a single site ( Nt 165, 171). Ctrl1: wild type Pneumocystis strain1, Ctrl2: wild type Pneumocystis strain2, Positive Ctrl3: submitted sulfa antibiotics resistance strain, *: Double test for Isolate 3 (after 2h incubation in 65°C in EDTA buffer)

Lactate dehydrogenase enzyme assay
The mean total LDH levels in co-infected patients (PCP in TB positive patients)was higher (445± 155 U/L) than enzyme level in patients with chronic TB (310±50 U/L). The difference was statistically significant using ANOVA analysis method(p<0.05). The ranges of LDH levels in the two groups are shown in table 1,in the control patients without PCP and TB LDH ranges was 77-240 U/L P < 0.05 (Fig. 3). The highest range of enzyme (601 IU/L) was reported in theserum and BAL samplesof patient with second Pneumocystis clinical isolate.

Fig. 3. Serum lactate dehydrogenase (LDH) in patients with Tuberculosis &Pneumocystis pneumonia co-infected cases in comparison to TB positive control group, LDH levels are shown for ten examples. There is obvious difference in enzyme value between two studied group that support aggravation of para clinical significance in Pneumocystis mix infection

DISCUSSION

Conformation sensitive gel electrophoresis (CSGE) is a rapid screening method for the detection of DNA sequence variation, specifically single-base changes or small insertions and deletions. It has been widely used for mutation screening in genetic disorders and for the detection of single nucleotide polymorphisms (SNPs)13. Detection of mutations in double-stranded DNA by gel electrophoresis is based on the supposition that a single-base mismatch can make conformational changes such as a bend in the double helix that causes differential migration of heteroduplexes and homoduplexes structures5. On electrophoresis in a non denaturing gel, heteroduplexes have retarded mobility compared to homoduplexes. The technique was first describedfor insertion/deletion mutations, but can also be applied to single-base replacement14. It was developed on the basis of the further assumption that mildly denaturing solvents in an appropriate buffer can accentuate the conformational changes produced by singlebase mismatches and thereby increase the differential migration of heteroduplexes and homoduplexes15.Under the initially described our conditions, 4 single-base mismatches were detected by CSGE in a series of PCR products in size 269bp and one sequence variations (No.3) that were not detected by CSGE was found.According toprevious studies, CSGE direct use in diversity identification of fungi is rare and the technique commonly has been used to detect mutations in human hereditary diseases16-17.  In a similar study about fungal diversity a modified PAGE system(HPA)be used that enables accurate identification of the species of AspergillusSection Flaviand subdivision based on highly sensitive discrimination of sequence variability7.They applied nucleotide sequence alignments, by using commercial panels to resulting heteroduplexes and found hydrolink mutation detection enhancement (MDE) gel (FMC BioProducts) in according the manufacturer’s instructions. while our resulting system was customary and manual. Conformation-sensitive gel electrophoresis (CSGE) is a variant of the HA method, employing mildly denaturing gel conditions. It is ideal for fragments differential  in size range of 200–800 bp, sensitivity of 88% has been detected recent developments in CSGE include the application of fluorescent labeling and detectionand capillary electrophoresis 14.In addition to the sensitivity, the advantage of CSGE over other molecular used techniques for scanning PCR products isno need to special equipment or preparation of PCR samples. EventuallyThe procedure is simple, requires little standardization, does not use radioactivity andthe standard polyacrylamide gel electrophoresis is used in a solvent buffer system.

Also we consider a hypothesis, significance of serum dehydrogenase indicator and mix morbidity of M.tuberculosisand Pneumocystis pneumonia.Total LDH level of >300 U/L should not be considered diagnostic; it should instead suggest that a diagnosis other than PJP or bacterial pneumonia be considered, even the patients with PJP seldom had LDH levels of>1,000 U/L 10. In addition, we did not assess the source of the excess LDH in our patients because we did not measure LDH isoenzyme levels. Therefore, we propose to evaluate LDH isoenzymes pattern (special LDH3) for next researches. The relevance of serial LDH measurements for the management of Pneumocystisclonization or for assessing the risk of relapse is an interesting question and deserves further studies18.As other investigations have confirmed this feature manifestations, elevated serum/BAL lactate dehydrogenase levels except low specificity have been noted in patients with PJP as high diagnostic value in novel clinical perspectives of Pneumocystosis.

ACKNOWLEDGMENTS

This study was financially supported by Research Deputy of TarbiatModares University.The authors wish to thank FirouzNouroozi for helpful technical assistance.

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