**Comparison of the MAKLER & HINRICHS (1993) Technique Versus Application of Hepes Lysis Solvent in Determining the Activities of**  *Plasmodium Lactate Dehydrogenase* **(pLDH) in**  *Plasmodium berghei***- Infected Erythrocytes**

Shafariatul A.I\* , Hasidah M. Sidek, Salmijah Surif

Department of Biomedical Science, Faculty of Allied Health Sciences, Universiti Kebangsaan, Kuala Lumpur, Malaysia

### **Abstract**

Plasmodium lactate dehydrogenase ( pLDH) is a clinically valuable diagnostic indicator for malaria disease. In this preliminary report, there are some considerable interest in measurements of pLDH assay. All these years, pLDH was evaluated according to method which was established by Makler and Hinrichs in 1993. This method has became very popular among malaria researches . In this study , we tried to describe an alternative way or method for measuring pLDH assay. The method was used Hepes lysis to disrupt the parasitized erythrocyte membrane and thus released the pLDH in the solution. Measurement was done based on the intensity of chromatographic changes in color through ELISA reader at 650nm. The Hepes lysis technique was recently introduced in few years ago. Both Makler (1993) and Hepes lysis techniques have demonstrated their ability and efficiency for assessing the pLDh assay . Based on the results, Makler and Hinrich ( 1993) technique have shown that pLDH assay activities were higher than Hepes lysis technique significantly ( 2 way ANOVA). The results were also supported by the microscopic view on both techniques .The experiment were conducted at 10% parasitemia and 30% parasitemia. Further investigation are needed in order to create and have more robust lactate sensing format and simple device with fast and precise respond.

**Keywords:** Plasmodium lactate dehydrogenase , Makler and Hinrich (1993), Hepes lysis , pLDH assay

## **1. Introduction**

*Plasmodium lactate dehydrogenase* (pLDH) is an essential metabolic enzyme that responsible for energy production in the parasite and parasite development. It is present in all malarial parasites of man and other animal host. pLDH catalyzes dehydrogenation of lactate and generates pyruvate by using NAD† as a cofactor. For more efficient and reliable test for pLDH activity, it can easily be differentiated from host lactate dehydrogenase (LDH) with 3-acetylpyridine adenine dinucleotide (APAD), an analogue of nicotinamide adenine dinucleotide (NAD) (Makler et. al 1993). pLDH can utilize APAD at 200 fold more rapidly and effectively than host LDH isoforms.

© 2012 Shafariatul et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The pLDH converts APAD† to APADH/H† and in turn reduces colorless Nitro Blue Tetrazolium (NBT) to blue formazan that regenerates APAD for another cycle of reaction with pLDH. It is apparent that this colormetric assay is very versatile, having the ability to follow drug resistance, monitoring drug therapy and diagnose malaria.

International Conference on Applied Life Sciences (ICALS2012)

C and thawed in 370 C water bath. Then all aliquots were transferred

The two techniques which are Makler et.al 1993 with some modifications and Hepes Lysis were

According to Makler et. al 1993, the blood samples which consisted of plasma or hemolyzed red blood cells would have gone through freeze and thaw cycle for at least 4 times. The blood

into 96- well, flat bottomed microtiter plate containing Malstat reagent ( 20mg of Sodium Llactate, 5.5mg of basic Tris Buffer ( Tris –C) and 3.7 mg of APAD dissolved in 1 ml of distilled water) and NBT-PES mixture (1.6mg of Nitroblue Tetrazolium ( NBT) and 0.1 mg of Phenazine ethosulphate (PES) dissolved in 1 ml of distilled water). Absorbance was measured at 650nm

lysed. The above procedures are directly assayed without incubation process. For incubation

For the technique that used Hepes Lysis, the blood samples were centrifuged at 10 000xg to obtain supernatant samples containing pLDH. The supernatants were then transferred into 96-well microtiter plate containing Malstat reagent and followed by addition of NBT-PES mixture. Then measurements of pLDH were assayed at 650nm using Elisa plate reader. As with the Makler

The min activities of pLDH for both techniques were analysed with 2 ways ANOVA test. Study had shown that activities of pLDH were significantly difference according to percentage level of

The pLDH activities by Makler technique (1.16 ) were higher than for the Hepes Lysis technique (0.800) ( Fig 1). Both are significantly different with F (1, 20) =27.042, (p<0.05). Infected cells with parasitemia at 10% were shown significantly different from normal cells or non parasitized cells (0% parasitemia) by both techniques . Based on Hepes lysis technique, the mean activity of pLDH in non parasitized cells (1.130 ± 0.031) were significantly lower than parasitized cells at 10% parasitemia(1.930 ± 0.027). The Makler technique gave lower mean activity of pLDH in non parasitized cells (1.560 ± 0.040) compared to parasitized cells at 10% parasitemia (2.72 ± 0.040). In comparison, the mean activity of pLDH in non parasitized cells ( 1.130 ± 0.031 ) and parasitized cells at 10% parasitemia (1.930 ± 0.027 ) were significantly lower by Hepes lysis technique than non parasitized ( 1.560 ± 0.040) and parasitized cells ( 10% parasitemia )( 2.721 ± 0.040) by Makler technique respectively . Briefly, the results from the above study demonstrated that Makler tech-

The above results were supported by the microscopic view on both techniques. . By doing freeze and thaw cycling, the red blood cells were almost completely lysed, whereas, Hepes Lysis still

were used whereas non parasitized red blood cells were prepared for the negative control.

C to lyse the red blood cells. For the positive control, parasitized red blood cells

**Plasmodium Lactate Dehydrogenase Bioassay**

samples were frozen at -20<sup>o</sup>

using an ELISA plate reader.

parasitemia and technique used.

cooled at -40<sup>o</sup>

**3. Results**

conducted to determine the activities of pLDH respectively.

In this study, the temperature for freezing was done at -400

process, the samples were incubated in a candle jar for 48h at 370

technique, some samples were incubated and some were directly assayed.

nique was more efficient and reliable than Hepes lysis technique.

Turkey, September 10-12, 2012

C for red blood cells to be completely

C, and were subsequently

<sup>279</sup> ISALS

The early method of assessing activity of pLDH was introduced by Makler et.al 1993. This technique has been widely used by many researchers with some modifications. The assay has been proven able to detect the presence of *Plasmodium falciparum* from *in vitro* cultures at parasitemia levels of 0.02% (Makler and Hinrichs, 1993 ). In recent years, there were some researchers working on pLDH by using Hepes lysis to hemolyzed red blood cells. The technique by While Makler et.al 1993 hemolyzed the red blood cells by four freeze-thaw cycles during which the samples were frozen at -20<sup>o</sup> C and thawed in a 37<sup>o</sup> C water bath. These two methods have different steps or procedures in order to obtained the pLDH and determined the formation of APADH at 650nm using a multiwavelength Elisa plate reader. Details of both techniques as explained in material and method section. The aim of this study is to compare the efficiency, precision, sensitivity and ease to evaluate pLDH activities between the two techniques. It is clear that both techniques hold a prominent position as the available sources of method to detect the presence of pLDH activities. It's also a reliable nonmicroscopic screening assay for any plasmodium species at a different level of sensitivity. Previous studies have showed a correlation between levels of parasitemia and the activity of parasite LDH (IMR, 2000; Vander Jagt et.al, 1981& Xu, X.L. et.al, 2007).

In this report, the *Plasmodium berghei* (*P. berghei* )– rodent model has been developed and used in this study to allow the determination of pLDH activities from both techniques. The significance of using malaria rodent instead of cultured human malaria is because it is easy and safe to handle and manipulate any stage of the life cycle in the laboratory (C.J.Janse, 1995). Hence, rodent malaria are still valuable models in several crucial areas of malaria research. The life cycle of *P.berghei* has similarities with *Plasmodium vivax* (*P.vivax*) which provide the first justification for the use of rodent in malaria research and definitely for in this study.

## **2. Material and method**

#### **Malarial Parasites**

The *Plasmodium berghei* ( NK-65) was obtained from swiss mice that reared in Universiti Kebangsaan Malaysia animal house. The *P. berghei* was used to test for the pLDH activities. Parasites are maintained thorough weekly blood passage in mice. The mice were treated and the experimental works were approval by Universiti Kebangsaan Malaysia Animal Ethics Committee (FSKB/ BIOMED/2009/ZAKIAH/20-AUGUST/275-AUGUST-2009-DECEMBER-2009).

#### **Detection of Parasitemia**

Parasitemia was determined by light microscopy (1000 x magnification) using Giemsa-stained thin smears. The percentage of parasitemia in mice was monitored until it reached up to required percentage parasitemia which are 10% and 30 %. Then the blood will be collected by orbital sinus or heart puncture. The collected blood was pooled in EDTA tube. Then it will readily to be used for further steps.

#### **Plasmodium Lactate Dehydrogenase Bioassay**

The two techniques which are Makler et.al 1993 with some modifications and Hepes Lysis were conducted to determine the activities of pLDH respectively.

According to Makler et. al 1993, the blood samples which consisted of plasma or hemolyzed red blood cells would have gone through freeze and thaw cycle for at least 4 times. The blood samples were frozen at -20<sup>o</sup> C and thawed in 370 C water bath. Then all aliquots were transferred into 96- well, flat bottomed microtiter plate containing Malstat reagent ( 20mg of Sodium Llactate, 5.5mg of basic Tris Buffer ( Tris –C) and 3.7 mg of APAD dissolved in 1 ml of distilled water) and NBT-PES mixture (1.6mg of Nitroblue Tetrazolium ( NBT) and 0.1 mg of Phenazine ethosulphate (PES) dissolved in 1 ml of distilled water). Absorbance was measured at 650nm using an ELISA plate reader.

In this study, the temperature for freezing was done at -400 C for red blood cells to be completely lysed. The above procedures are directly assayed without incubation process. For incubation process, the samples were incubated in a candle jar for 48h at 370 C, and were subsequently cooled at -40<sup>o</sup> C to lyse the red blood cells. For the positive control, parasitized red blood cells were used whereas non parasitized red blood cells were prepared for the negative control.

For the technique that used Hepes Lysis, the blood samples were centrifuged at 10 000xg to obtain supernatant samples containing pLDH. The supernatants were then transferred into 96-well microtiter plate containing Malstat reagent and followed by addition of NBT-PES mixture. Then measurements of pLDH were assayed at 650nm using Elisa plate reader. As with the Makler technique, some samples were incubated and some were directly assayed.

#### **3. Results**

The min activities of pLDH for both techniques were analysed with 2 ways ANOVA test. Study had shown that activities of pLDH were significantly difference according to percentage level of parasitemia and technique used.

The pLDH activities by Makler technique (1.16 ) were higher than for the Hepes Lysis technique (0.800) ( Fig 1). Both are significantly different with F (1, 20) =27.042, (p<0.05). Infected cells with parasitemia at 10% were shown significantly different from normal cells or non parasitized cells (0% parasitemia) by both techniques . Based on Hepes lysis technique, the mean activity of pLDH in non parasitized cells (1.130 ± 0.031) were significantly lower than parasitized cells at 10% parasitemia(1.930 ± 0.027). The Makler technique gave lower mean activity of pLDH in non parasitized cells (1.560 ± 0.040) compared to parasitized cells at 10% parasitemia (2.72 ± 0.040). In comparison, the mean activity of pLDH in non parasitized cells ( 1.130 ± 0.031 ) and parasitized cells at 10% parasitemia (1.930 ± 0.027 ) were significantly lower by Hepes lysis technique than non parasitized ( 1.560 ± 0.040) and parasitized cells ( 10% parasitemia )( 2.721 ± 0.040) by Makler technique respectively . Briefly, the results from the above study demonstrated that Makler technique was more efficient and reliable than Hepes lysis technique.

The above results were supported by the microscopic view on both techniques. . By doing freeze and thaw cycling, the red blood cells were almost completely lysed, whereas, Hepes Lysis still showed some red blood cells that did not lyse. The hemolysed parasitized cells will release the pLDH in homogenates or supernatant. The more the cells lysed the bigger chance of parasitized cells to be lysed as well.

International Conference on Applied Life Sciences (ICALS2012)

NADH or NADPH. In *Plasmodium*, the lactate dehydrogenase works efficiently 200 times higher

The assay development was introduced and established by Makler and Hinrichs in 1993. For many years, the assay was used in diagnosing malaria infection. In recent years, it has become popular as a validated target for antimalarial agent development in drug screening. In the meantime, there is an alternative method of pLDH assay by using Hepes Lysis solvent. It was used by some researchers but the performance is still under investigation. Basically, the principal for both methods were lysed the membrane erythrocyte. By doing so, pLDH would be released and allow to be measured by Elisa reader based on colorimetric changes. From these studies both methods have shown significant results. The negative control which was normal cells or non parasitized cells showed lower activities. However, the findings showed that Makler and

According to Makler and Hinrichs technique (1993) , thaw and freeze cycle process will disrupted the structure of erythrocyte membrane . Disruption will lead to cells hemolysate . For parasitized erythrocyte , hemolysate process assisted in releasing the pLDH. The more the parasitized cells lysed , the more the pLDH would released . The lysate erythrocyte turned out to be small

By using Hepes lysis solvent , erythrocyte was lysed chemically. Hepes lysis was alkaline solution with pH 8. For normal cells the pH ranging between 7.34 to 7.45. Zeidler and Kim (1977) , reported that alkaline condition will disrupt the stability of the cell membrane protein and eventually lysed. Most of the erythrocytes were not lysed. The morphology of treated erythrocyte were seen similar as in control cells or untreated cells. Probably the Hepes lysis need to be more

Based on the overall results, erythrocytes that were treated with Makler and Hinrichs technique (1993) showed significantly higher activities than erythrocytes treated with Hepes Lysis. The results were supported by microscopic view .The activities of pLDH will increased in relation to the higher numbers of parasites. Comparison of pLDH activities were conducted by Makler and Hinrichs ( 1993 ) technique at 30% and 10% parasitemia respectively .The parasitized erythrocytes at 30% parasitemia presented significantly higher pLDH activity than 10% parasitemia

Regarding the morphology of erythrocyte infected-*Plasmodium berghei* , the erythrocytes as the host may had more than one parasites reside in it. The size of the infected erythrocytes is bigger than normal cells. The features were similar to *Plasmodium vivax* .The colour were light purplish red. The Plasmodium could be found in various stages, mostly, were ring and trofozoite form. On the other hand , the pLDH assay showed higher activities in cells without incubation process compared to cells treated by incubation process. This might be due to the contamination of the

In this study, Makler and Hinrichs (1993 ) technique showed to be more efficient than Hepes lysis

than human lactate dehydrogenase with APAD.

debris and scattered in it's surrounding.

alkaline to work efficiently.

and 0% parasitemia respectively.

**5. Summary and conclusion**

cells which occured during the incubation period.

solvent to quantitate the activities of *Plasmodium Lactate dehydrogenase* .

Hinrichs (1993) technique exhibited higher activities than Hepes Lysis.

Turkey, September 10-12, 2012

<sup>281</sup> ISALS

In this study, densities of infected cells do play a major role to achieve high activity of pLDH assay. As we have seen, the higher parasitemia , the more infected cells containing pLDH to be released. The 30% parasitemia indicated that 30 out of 100 red blood cells were infected with plasmodium .Likewise, 10% parasitemia indicated that 10 of 100 red blood cells were infected.

**Fig 1.** Determination Mean Activities of pLDH Between Makler ( 1993) Technique And Hepes Lysis

a = Min activity of pLDH in normal cells ( 0% parasitemia ) which are significantly different compared to infected cells ( 10% parasitemia ) by Hepes lysis technique.

b = Min activity of pLDH in normal cells ( 0% parasitemia ) which are significantly different compared to infected cells ( 10% parasitemia ) by Makler technique.

c = Min activity of pLDH in normal cells ( 0% parasitemia ) by Hepes lysis technique which are significantly lower compared to min activity of pLDH in normal cells ( 0% parasitemia ) by Makler technique .

d= Min activity of pLDH in infected cells (10% parasitemia ) by Hepes lysis technique which are significantly lower compared to min activity of pLDH in infected cells (10% parasitemia ) by Makler technique .

#### **4. Discussion**

Initially pLDH assay was developed to detect the presence of Plasmodium in the erythrocytes of malaria patient. The assay uses APAD (3-acetyl pyridine adenine dinucleotide ) which is an analog to NAD ( β- Nicotinamide adenine dinucleotide ) with higher oxidation potential than NAD. It can substitute for NAD as a hydrogen accepting cofactor in many dehydrogenase reactions ; e.g : lactate dehydrogenase from *Toxoplasma* , *Clonorchis* , *Plasmodium* as well as mammalian dehydrogenase. It can also act as a proton acceptor in various transhydrogenation reactions with

NADH or NADPH. In *Plasmodium*, the lactate dehydrogenase works efficiently 200 times higher than human lactate dehydrogenase with APAD.

The assay development was introduced and established by Makler and Hinrichs in 1993. For many years, the assay was used in diagnosing malaria infection. In recent years, it has become popular as a validated target for antimalarial agent development in drug screening. In the meantime, there is an alternative method of pLDH assay by using Hepes Lysis solvent. It was used by some researchers but the performance is still under investigation. Basically, the principal for both methods were lysed the membrane erythrocyte. By doing so, pLDH would be released and allow to be measured by Elisa reader based on colorimetric changes. From these studies both methods have shown significant results. The negative control which was normal cells or non parasitized cells showed lower activities. However, the findings showed that Makler and Hinrichs (1993) technique exhibited higher activities than Hepes Lysis.

According to Makler and Hinrichs technique (1993) , thaw and freeze cycle process will disrupted the structure of erythrocyte membrane . Disruption will lead to cells hemolysate . For parasitized erythrocyte , hemolysate process assisted in releasing the pLDH. The more the parasitized cells lysed , the more the pLDH would released . The lysate erythrocyte turned out to be small debris and scattered in it's surrounding.

By using Hepes lysis solvent , erythrocyte was lysed chemically. Hepes lysis was alkaline solution with pH 8. For normal cells the pH ranging between 7.34 to 7.45. Zeidler and Kim (1977) , reported that alkaline condition will disrupt the stability of the cell membrane protein and eventually lysed. Most of the erythrocytes were not lysed. The morphology of treated erythrocyte were seen similar as in control cells or untreated cells. Probably the Hepes lysis need to be more alkaline to work efficiently.

Based on the overall results, erythrocytes that were treated with Makler and Hinrichs technique (1993) showed significantly higher activities than erythrocytes treated with Hepes Lysis. The results were supported by microscopic view .The activities of pLDH will increased in relation to the higher numbers of parasites. Comparison of pLDH activities were conducted by Makler and Hinrichs ( 1993 ) technique at 30% and 10% parasitemia respectively .The parasitized erythrocytes at 30% parasitemia presented significantly higher pLDH activity than 10% parasitemia and 0% parasitemia respectively.

Regarding the morphology of erythrocyte infected-*Plasmodium berghei* , the erythrocytes as the host may had more than one parasites reside in it. The size of the infected erythrocytes is bigger than normal cells. The features were similar to *Plasmodium vivax* .The colour were light purplish red. The Plasmodium could be found in various stages, mostly, were ring and trofozoite form.

On the other hand , the pLDH assay showed higher activities in cells without incubation process compared to cells treated by incubation process. This might be due to the contamination of the cells which occured during the incubation period.

## **5. Summary and conclusion**

In this study, Makler and Hinrichs (1993 ) technique showed to be more efficient than Hepes lysis solvent to quantitate the activities of *Plasmodium Lactate dehydrogenase* .

## **6. Acknowledgement**

I would like to express my greatest gratitude to Faculty of Science and Technology, UKM and supportive staffs from the Department of Biomedical science, Faculty of Allied Health Sciences UKM.

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,

, Nik Marzuki Sidik<sup>4</sup>

**Detection of Rifampin- and Isoniazid-Resistant** 

**Genes in** *Mycobacterium tuberculosis* **Clinical** 

1 School of Diagnostic and Applied Health, Faculty of Health Sciences, Universiti Kebangsaan

3 Bacteriology Unit, Infectious Disease Research Centre, Institute of Medical Research, Kuala Lumpur 4 School of Biosciences and Biotechnology, Faculty of Science and Technology, University

Corresponding author. Tel.: +603 9287 7373; fax: +603 26938719, E-mail: nora@medic.ukm.my

Tuberculosis still remains the leading cause of death worldwide. The morbidity has been reported to decrease, but incident and prevalence of multi-drug resistance Tuberculosis is still on rise. Rifampicin and isoniazid are the first line treatment to TB patients and resistance to these drugs has been linked to mutations in genes such as *rpoB* and k*atG*. In the present study, PCR method was employed to detect three types of restricted genes associated with drug resistance tuberculosis namely *rpoB*, regulatory-*inhA* and *katG*. Sixty-two samples were obtained from different parts of Malaysia hospital which consisted of 35 pulmonary and 27 extra-pulmonary specimens. Twentyseven specimens showed positive results as detected by duplex PCR method whereas 3 specimens positive as detected by acid fast bacilli and culture method. Out of 27 isolates, 3 isolates from culturable isolates harbored restricted genes that where associated with drug resistance tuberculosis. The mutations involved in *rpoB* genes comprised of acid amino transposition (isolate 148) and frameshift mutations (isolate 624 and 374). This study is clinically important because it focuses in molecular diagnosis and can act as an early warning on the emerging status of multidrug resis-

Tuberculosis (TB) is an infectious bacterial disease caused by *Mycobacterium tuberculosis* which commonly affects the lungs and respiratory system. The TB morbidity was reported decreasing, but its remains alarming due to increase in incidence and prevalence of Multi Drug Resistance-TB (MDR-TB) [1]. To date, seven antimicrobial agents that have been used in the treatment of resistant TB cases were; - isoniazid, rifampicin, pyraxinamide, enthambutol, streptomycin and fluoroqui-

Noraziah Mohamad Zin1, \*, Nor Farha Hussain<sup>2</sup>

2 Pathology Unit, Hospital of Tengku Anis, Pasir Puteh, Kelantan

, Mohamed Kamel A Ghani<sup>1</sup>

**Isolates**

Rahizan Isa<sup>3</sup>

\*

**Abstract**

Malaysia, Kuala Lumpur

Kebangsaan Malaysia, Bangi Selangor

tance of *M. tuberculosis* in Malaysia.

**1. Introduction**

© 2012 Zin et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the

original work is properly cited.

**Keywords**: Rifampin, Isoniazid-Resistant, M. tuberculosis, PCR

Turkey, September 10-12, 2012

<sup>283</sup> ISALS

#### **7. References**


## **Detection of Rifampin- and Isoniazid-Resistant Genes in** *Mycobacterium tuberculosis* **Clinical Isolates**

Noraziah Mohamad Zin1, \*, Nor Farha Hussain<sup>2</sup> ,

Rahizan Isa<sup>3</sup> , Mohamed Kamel A Ghani<sup>1</sup> , Nik Marzuki Sidik<sup>4</sup>

1 School of Diagnostic and Applied Health, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Kuala Lumpur

2 Pathology Unit, Hospital of Tengku Anis, Pasir Puteh, Kelantan

3 Bacteriology Unit, Infectious Disease Research Centre, Institute of Medical Research, Kuala Lumpur

4 School of Biosciences and Biotechnology, Faculty of Science and Technology, University Kebangsaan Malaysia, Bangi Selangor

\* Corresponding author. Tel.: +603 9287 7373; fax: +603 26938719, E-mail: nora@medic.ukm.my

#### **Abstract**

Tuberculosis still remains the leading cause of death worldwide. The morbidity has been reported to decrease, but incident and prevalence of multi-drug resistance Tuberculosis is still on rise. Rifampicin and isoniazid are the first line treatment to TB patients and resistance to these drugs has been linked to mutations in genes such as *rpoB* and k*atG*. In the present study, PCR method was employed to detect three types of restricted genes associated with drug resistance tuberculosis namely *rpoB*, regulatory-*inhA* and *katG*. Sixty-two samples were obtained from different parts of Malaysia hospital which consisted of 35 pulmonary and 27 extra-pulmonary specimens. Twentyseven specimens showed positive results as detected by duplex PCR method whereas 3 specimens positive as detected by acid fast bacilli and culture method. Out of 27 isolates, 3 isolates from culturable isolates harbored restricted genes that where associated with drug resistance tuberculosis. The mutations involved in *rpoB* genes comprised of acid amino transposition (isolate 148) and frameshift mutations (isolate 624 and 374). This study is clinically important because it focuses in molecular diagnosis and can act as an early warning on the emerging status of multidrug resistance of *M. tuberculosis* in Malaysia.

**Keywords**: Rifampin, Isoniazid-Resistant, M. tuberculosis, PCR

## **1. Introduction**

Tuberculosis (TB) is an infectious bacterial disease caused by *Mycobacterium tuberculosis* which commonly affects the lungs and respiratory system. The TB morbidity was reported decreasing, but its remains alarming due to increase in incidence and prevalence of Multi Drug Resistance-TB (MDR-TB) [1]. To date, seven antimicrobial agents that have been used in the treatment of resistant TB cases were; - isoniazid, rifampicin, pyraxinamide, enthambutol, streptomycin and fluoroqui-

© 2012 Zin et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

nolones. MDR phenotype is defined as resistance at least to isoniazid and rifampicin which is the

most effective drug recommended by World Health Organization (WHO) and being used as the first line treatment in TB patient. Therefore, resistance towards these two drugs has become major problems in the treatment of TB patients. Resistance to first line anti-TB drugs has been linked to mutations in at least 10 genes; *katG, inhA, ahpC, kasA* and *ndh* for INH resistance; *rpoB* for RIF resistance, *embB* for EMB resistance, *pncA* for PZA resistance and *rpsL* and *rrs* for STR resistance [2]. Nevertheless, nearly 95% of the RIF resistant strains possess a mutation in the *rpoB* gene encoding a DNA-dependent RNA polymerase [3]. In addition, approximately 90% of INH resistant strains have a mutation in the *inhA*, *katG*, and *ahpG* genes encoding enzymes that are related to mycolic acid synthesis of cell wall [3]. Rapid development of drug resistance caused by *M. tuberculosis* has led to measure resistance accurately and easily. This knowledge will certainly help us to understand how to prevent the occurrence of drug resistance as well as identifying genes associated with new drug resistance. Keeping the above facts in mind, the present, this study was carried out to detect resistance-associated mutations gene in *M. tuberculosis* clinical isolates.

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at 95 ºC for 30 seconds, annealing for 30 seconds at 64, 63, and 65 ºC for *inhA, rpoB* and *katG*  respectively.The extension cycle were72 ºC for 90 seconds for *inhA* and *katG* genes whereas 60 seconds for *rpoB* genes. The final extension was carried out at 72 ºC for 5 min. The PCR product was analysed by a 1.6 % agarose gel. DNA fragments of PCR products were purified using QIAquick PCR Purification Kit (QIAGEN, Germany) according to manufacturer's recomendation. Purified DNA fragments were sent to the 1st Base Laboratories Sdn. Bhd, Petaling Jaya, Kuala Lumpur for sequencing. DNA sequences were analyzed using BLAST (http://www.ncbi.nlm. nih.gov/BLAST). Multiple sequence alignments were conducted using ClustalW (http://www.

In total, 27 extra-pulmonary and 35 pulmonary clinical specimens were obtained for AFB smear, culture and PCR analysis. Out of 62 isolates, only 3 isolates namely 148, 374 and 624 were culture positive as detected by AFB. The culture was then confirmed as *M. tuberculosis complex* using GenProbe System performed by Bacteriology Unit, National Public Health Laboratory at Sungai Buloh, Kuala Lumpur. Nevertheless 27 isolates were positive for IS6110 of *M. tuberculosis* as detected by DPCR. Of the 27 isolates, 12 of them were from extra-pulmonary isolates and 15 were from pulmonary isolates (Figure 1). It was interesting to note that the PCR methods were able to detect the AFB negative of the non-pulmonary specimens such as from CSF, bone biopsy, lumbar puncture, rectal biopsy and lymph nodes. A similar result was shown by Suhaila et al. (2008) [4]. Number of positive isolates detected by PCR analysis was higher compared to culture method. Time required for detection of *M. tuberculosis* from clinical specimens via PCR analysis was less

Out of 27 isolates positive for *M. tuberculosis* genes, only 3 culturable isolates showed amplification of all resistance genes tested (Figure 2). Low quantity of *M. tuberculosis* DNA in the clinical sample and the presence of inhibitor [5] might be the contributed factors. It was interesting to note that all the positive samples bearing the Zielhl Neelson staining scores more that 10/3L directly showed the high bacteria numbers. Approximately 104 organisms/ml are required for reliable detection with Ziehl-Neelsen stains [6] In addition, the properties of the genes of interest also affect the success of amplification. Only one specific site of *katG*, *rpoB* and regulatory-*inhA*  genes presence in the genome of *M. tuberculosis* and may explain the limited excess to the genes. Hence, the amplification cannot be made. The primer used for detection of resistance genes have been successfully design by Vector NTI software. Amplification of resistance genes from clinical specimen have been succeeding in three isolates namely 148, 374 and 624. Amplified genes for *rpoB*, *katG* and *inhA* were shown in Figure 2a, 2b and 2c respectively. The product size for *rpoB, katG* and *inhA* were 442bp, 2206bp and 442bp, respectively as confirmed by sequencing analysis.

**3.1. Detection of M. tuberculosis by PCR and culture method** 

than 4 hours whereas more than 8 weeks were required in culture method.

genome.jp/tools/clustalw/).

**3. Result and Discussion**

**3.2. Detection of resistance genes** 

Turkey, September 10-12, 2012

<sup>285</sup> ISALS

## **2. Materials and methods**

#### **2.1. Bacterial isolates and clinical samples**

Bacterial samples used in this study were collected from various hospitals in Malaysia. Prior ethical approval was obtained from the institutional ethics committee. The samples consisted of pulmonary (n=35) and non-pulmonary (n=27) specimens. The samples such as sputum, gastric lavage and urine were decontaminated with 4% NaOH for 15 minutes before being used. All samples were investigated for the presence of acid-fast bacilli by Ziehl-Neelsen and cultured on Loewenstein-Jensen medium. *M. tuberculosis* strain ATCC 27294 was grown in Ogawa medium and used as positive control. Whereas, *Bacillus subtilis* (ATCC 26633) was grown on blood agar and used as negative control.

## **2.2. DNA extraction, PCR amplifications and DNA sequencing analysis**

Extraction of DNA from bacterial culture and clinical samples were carried out by using High Pure Viral Nucleic Acid Extraction Kit (Roche Inc. USA) according to manufacturer's recommendation. DNA from all bacterial isolates and clinical samples were subjected to Duplex-PCR using mixture of primers to amplify the IS*6110* gene and p*53* gene as previous report [4] with some modifications. The amplification mixture consisted of 1 µg of template DNA, 1X final concentration of MasterMix (Eppendorf), and primers (0.4 pmole/µl). Amplification was carried out using Master Cycler Gradient Thermocycler (Eppendorf). The cycling parameters were 94 ºC for 3 min followed by 34 cycles of denaturation at 94 ºC for 15 seconds, annealing at 66 ºC for 15 seconds and extension at 72 ºC for 20 seconds. Final extension was then carried out at 72 ºC for 3 min. The PCR product was analysed by a 1.6 % agarose gel. Primers used for detection of *rpo*B, *inh*A and *kat*G resistance genes were design by Vector NTI software. The amplification mixture consisted of 1 µg of template DNA, 1X final concentration of MasterMix (Eppendorf), and primers (0.4 pmole/µl). The amplification was carried out using Master Cycler Gradient Thermocycler (Eppendorf). The cycling parameters were 95 ºC for 3 min followed by 34 cycles of denaturation at 95 ºC for 30 seconds, annealing for 30 seconds at 64, 63, and 65 ºC for *inhA, rpoB* and *katG*  respectively.The extension cycle were72 ºC for 90 seconds for *inhA* and *katG* genes whereas 60 seconds for *rpoB* genes. The final extension was carried out at 72 ºC for 5 min. The PCR product was analysed by a 1.6 % agarose gel. DNA fragments of PCR products were purified using QIAquick PCR Purification Kit (QIAGEN, Germany) according to manufacturer's recomendation. Purified DNA fragments were sent to the 1st Base Laboratories Sdn. Bhd, Petaling Jaya, Kuala Lumpur for sequencing. DNA sequences were analyzed using BLAST (http://www.ncbi.nlm. nih.gov/BLAST). Multiple sequence alignments were conducted using ClustalW (http://www. genome.jp/tools/clustalw/).

## **3. Result and Discussion**

#### **3.1. Detection of M. tuberculosis by PCR and culture method**

In total, 27 extra-pulmonary and 35 pulmonary clinical specimens were obtained for AFB smear, culture and PCR analysis. Out of 62 isolates, only 3 isolates namely 148, 374 and 624 were culture positive as detected by AFB. The culture was then confirmed as *M. tuberculosis complex* using GenProbe System performed by Bacteriology Unit, National Public Health Laboratory at Sungai Buloh, Kuala Lumpur. Nevertheless 27 isolates were positive for IS6110 of *M. tuberculosis* as detected by DPCR. Of the 27 isolates, 12 of them were from extra-pulmonary isolates and 15 were from pulmonary isolates (Figure 1). It was interesting to note that the PCR methods were able to detect the AFB negative of the non-pulmonary specimens such as from CSF, bone biopsy, lumbar puncture, rectal biopsy and lymph nodes. A similar result was shown by Suhaila et al. (2008) [4]. Number of positive isolates detected by PCR analysis was higher compared to culture method. Time required for detection of *M. tuberculosis* from clinical specimens via PCR analysis was less than 4 hours whereas more than 8 weeks were required in culture method.

#### **3.2. Detection of resistance genes**

Out of 27 isolates positive for *M. tuberculosis* genes, only 3 culturable isolates showed amplification of all resistance genes tested (Figure 2). Low quantity of *M. tuberculosis* DNA in the clinical sample and the presence of inhibitor [5] might be the contributed factors. It was interesting to note that all the positive samples bearing the Zielhl Neelson staining scores more that 10/3L directly showed the high bacteria numbers. Approximately 104 organisms/ml are required for reliable detection with Ziehl-Neelsen stains [6] In addition, the properties of the genes of interest also affect the success of amplification. Only one specific site of *katG*, *rpoB* and regulatory-*inhA*  genes presence in the genome of *M. tuberculosis* and may explain the limited excess to the genes. Hence, the amplification cannot be made. The primer used for detection of resistance genes have been successfully design by Vector NTI software. Amplification of resistance genes from clinical specimen have been succeeding in three isolates namely 148, 374 and 624. Amplified genes for *rpoB*, *katG* and *inhA* were shown in Figure 2a, 2b and 2c respectively. The product size for *rpoB, katG* and *inhA* were 442bp, 2206bp and 442bp, respectively as confirmed by sequencing analysis.

International Conference on Applied Life Sciences (ICALS2012)

isolate 148 showed minor mutation where the additional, changes and transposition of certain base of amino acid Whereas, isolate 374 showed showed frameshift mutation in which total loss of A base that subsequently affects the rest of amino acid coding. It is known as.. Nevertheless, isolate 624 showed both minor and major mutation at different base position in which all amino

Two isolates of 624 and 374 showed frameshift mutations in which all the amino acid codes were totally changed. the quantity of DNA and the present of inhibitor may affect the successful of amplification. In addition, only one specific site of *katG*, *rpoB* and *inhA* sequence in whole genome of *M. tuberculosis.*. As for conclusion*,* the identification of *M. tuberculosis* through amplification of *IS6110* was successfully achieved whereas amplification of restricted genes associated with drug

This research study was funded by Ministry of higher education of Malaysia UKM-NN-07-

[1] WHO, 2008. Global Tuberculosis Control 2008, Surveillance,Planning, Financing. *WHO Regional* 

[2] Johnson R., Streicher E.M., Louw G.E., Warren R.M. 2008. Drug Resistance in *M. tuberculosis*.

[3] Sajduda, A., Brzostek A., Popławska M., Augustynowicz-Kopec E., Zwolska Z., Niemann S., Dziadek J., and Hillemann D. 2004. Molecular Characterization of Rifampin- and Isoniazid-Resistant *Mycobacterium tuberculosis s*trains Isolated in Poland. *Journal of Clinical Microbiology*.

[4] Suhaila, H., Rahizan, I., Nik Marzuki S. and Noraziah M. Zin, 2008. The Usefulness of PCR Amplification for Direct Detection of *M. tuberculosis* DNA from Clinical Samples. *Biotechnology* 

[5] Leven, M. and Goossens,H. 1997.Relevance of nucleic acid amplification techniques for diagnostis of respiratory transinfections in the clinical laboratory. *Clinical Microbiology Review*.10:242 [6] Wilson, M.L, Stone, B.L and Hildred, M.V. 1995. Comparison of recovery rates of mycobacteria from BACTEC 12B vials, Middlebrook 7H11 selective biplates and Lowenstein-Jensen slants in a public health mycobacteriology laboratory. Journal of Clinical Microbiology 33: 2516-2518

FRGS0224-2010 and UKM Research Grant University UKM-OUP-TKP-21-99/2009

acid code was totally changed.

resistance was achieved in culturable clinical specimens.

**4. Conclusion** 

**5. Acknowledgment**

42(6): 2425–2431

7(1): 100-105

*Office For the Western Pacific* 

Curr. Issues Mol. Biol. 8: 97–112

**6. References** 

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**Figure 1.** Agarose gel showing amplification of p53 and ISP6110. Amplifications were performed using marker 100 bp (M) and DNA from M. tuberculosis ATCC 27294 (P), various isolate (1-5), negative control, B. subtilis (N), positive control (PP).

**Figure 2.** Amplification of rpoB gene (a), katG gene (b) and inhA gene (c) From left; Lane HL: Hyperladder maker, Lane 1, isolate 148; Lane 2, isolate 374; Lane 3, isolate 624; Lane B, negative control, Lane P, positive controlMutation analysis

PCR products amplified from regulatory-*inhA*, *rpoB* and *katG* genes of *M. tuberculosis* were sequenced with the same primer used in amplification of the genes. The sequences obtained were compared to the sequences available in Gen Bank Data using BlastN analysis. The DNA nucleotide sequences were translated to amino acid sequences through BlastX software available in http://www.ncbi.nlm.nih.gov. The nucleotide and amino acid changes of each isolates for respective genes were compared using ClustalW (Vector NTI). As for mutation analysis of *rpoB* genes, isolate 148 showed minor mutation where the additional, changes and transposition of certain base of amino acid Whereas, isolate 374 showed showed frameshift mutation in which total loss of A base that subsequently affects the rest of amino acid coding. It is known as.. Nevertheless, isolate 624 showed both minor and major mutation at different base position in which all amino acid code was totally changed.

## **4. Conclusion**

Two isolates of 624 and 374 showed frameshift mutations in which all the amino acid codes were totally changed. the quantity of DNA and the present of inhibitor may affect the successful of amplification. In addition, only one specific site of *katG*, *rpoB* and *inhA* sequence in whole genome of *M. tuberculosis.*. As for conclusion*,* the identification of *M. tuberculosis* through amplification of *IS6110* was successfully achieved whereas amplification of restricted genes associated with drug resistance was achieved in culturable clinical specimens.

## **5. Acknowledgment**

This research study was funded by Ministry of higher education of Malaysia UKM-NN-07- FRGS0224-2010 and UKM Research Grant University UKM-OUP-TKP-21-99/2009

#### **6. References**


International Conference on Applied Life Sciences (ICALS2012)

**Contribution to the Study of the Impact** 

**of Phosphate Fertilizer on Biochemical** 

, Djebar M. R

Laboratory of Toxicology and cellular bioenergetics, University of Annaba, Algeria

In this study we are interested in assessing the impact of different regimes of NPK and its effect on wheat Triticum durum. The first results show that the presence of fertilizer causes a decrease in the percentage and speed of germination and an inhibition of growth of wheat. On the metabolic level, the NPK caused a significant increase in mean levels of proline and soluble

Adequate fertilization is a prerequisite for modern agriculture in order to meet high yields and optimum quality of crops. The vast majority of mineral elements essential to plant development, is only necessary in minute amounts, which may be provided by most soils without supplements. However, some key elements that are phosphorus (P), potassium (K), sulfur (S) and nitrogen (N), are frequently in short supply in the soil to allow optimal growth of crops. Despite its abundance, atmospheric nitrogen is unusable for most plants. The only plants able to use nitrogen from the air are those that develop a symbiotic relationship with microorganisms in nodules on plant roots. Unlike phosphorus and potassium, plants need large amounts of nitrogen, nitrogen representing 3-4% of their dry matter. Moreover the efficiency of nitrogen fertilization on crop yields contributed to the development of modern agriculture more intensive nitrogen. In addition to heavy loads caused by this item of expenditure, the massive inflow of nitrogen fertilizers started in the middle of last century affects the natural nitrogen cycle, which is not without consequences for

The objective of this study is to evaluate the effects of NPK fertilizer nitrogen on physiological

The experimental material used in our work is wheat (Triticum durum) Geta come from hard

parameters, biochemical and metabolic ones on wheat *Triticum durum*.

JTGC (demonstration farm and seed production of Guelma).

**Parameters of** *Triticum durum*

\*Corresponding author, Email: Sabrina\_bouchelaghem@yahoo.fr

**Keywords**: NPK, Proline, Protein, Soluble sugar, *Triticum durum*.

Sabrina Bouchelaghem\*

sugars, and inhibition of protein synthesis.

**Abstract**

**1. Introduction**

the environment and health.

**2. Materials and methods**

**2.1. Biological material**

© 2012 Bouchelaghem et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

provided the original work is properly cited.

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