Journal Of Health Science Vol IX.
No.
2 2024 | 6-12
Nucleic Acid Amplification Test In Pulmonary Diseases Fariz Nurwidya1,*.
Asri Liqditta Bies1.
Wira Winardi2.
Fadlika Harinda1 1Department of Pulmonology and Respiratory Medicine.
Faculty of Medicine.
Universitas Indonesia Ae Persahabatan Hospital.
Jakarta.
Indonesia.
2Department of Respiratory Medicine.
Juntendo University Graduate School of Medicine.
Tokyo.
Japan.
Email : fariz.
nurwidya@ui.
id , biesasriliqdita@gmail.
com, w-winardi@juntendo.
harindafadlika@gmail.
Corresponde Author: fariz.
nurwidya@ui.
Article Information Article History Received: 12-08-24 Revised: 15-09-24 Accepted:15-10-24 Keywords:
Nucleic Acid Amplification Test Pulmonary Diseases ABSTRACT Nuclei as a cell nucleus have been found since the 18 th century.
This discovery was then developed for the need for methods for diagnosing pathogenic organisms of an infectious disease at that time.
The aim of this study is to review the principle of nucleic acid amplification test and describe the existing evidence regarding the role of the test in respiratory The study use the keywords, such as nucleic acid, diagonsis and nucleic acid amplification test, to invesitage relevant studies in PubMed and Google Scholar database.
We found that nucleic acid-based testing techniques have evolved to adapt to the need for early and rapid detection and rely on a high level of accuracy.
This continuously developed technology can replace conventional test methods.
Conventional examination is known to take longer time so the definitive therapeutic might be delayed.
However, there is still an obstacle, namely the cost of tests quite expensive because of the standard procedure that must be strictly followed, otherwise result might not be valid.
The technique from the beginning needs to be carried out following the procedure, starting with the preparation of tools, how to take samples, how to deliver the samples to the laboratory, and how to process and analyze data.
This procedure is necessary to minimize false positive or negative results.
As a conclusion, nucleic acid assays is beneficial in pulmonary infectious diseases diagnosis, however it is high-cost and not recommended for treatment evaluation in pulmonary infection.
INTRODUCTION
Friedrich Miescher identified the nucleic acids in the year of 1868 using an isolation technique from the leucocytes.
The molecules were later known as A nucleic acid macromolecule made up of many smaller molecular units.
6 Journal Of Health Science These small units called nucleotides are chemically linked together in a chain (Csako, 2.
Deoxyribonucleic acid (DNA) is a molecular unit consist of four types of small size molecules described as nucleotide bases, namely cytosine (C), adenine (A), thymine (T), and guanine (G).
Email : jurnalfik@wiraraja.
Fariz et al.
Nucleic Acid Amplification Test InA.
DNA polymerase is an enzyme capable of building new copies of DNA in the shape of nucleic acid Scientists have utilized DNA polymerase through a Polymerase Chain Reaction (PCR) assay (Stellato et al.
, 2.
POLYMERASE CHAIN REACTION (PCR)
The beginning of the development of PCR examination was the discovery of thermostable polymerized DNA extracted from a bacterium that grows in hot springs called Thermus aquaticus.
This polymerase is resistant to the heating and cooling required in PCR assays and allows PCR examination requires four main They are nucleotide triphosphate.
DNApolymerase that is stable in various temperature, gene-specific primers, and the target DNA for The source DNA is obtained by isolating genomic DNA from the cell samples and tissues or performing ribonucleic acid (RNA) materials reverse transcription (Jocefczuk et al.
, 2.
The first step of PCR examination is separating the double strands .
sDNA) by incrasing the temperature of specimen material to 900 C.
The second is making base-specific complementary DNA strands by cooling.
The third is heating the mixed sample material until it reaches a temperature of 720 C.
This temperature is the best temperature for Taq DNA polymerase process (Wang et al.
, 2.
Based on DNA amplification and manipulation.
PCR assays extend molecular biology investigations.
The desired genome is then subject to amplification from genomic DNA and incorporated in plasmids or other vectors for further study.
PCR assay is known to have a role in diagnosing diseases.
PCR enables gene mutations identification by genome sequencing.
Thus, it helps to recognize diseases such as cancer (Pavsic et al.
, 2.
THE
ROLE
NUCLEIC
ACID-BASED
EXAMINATIONS FOR TUBERCULOSIS DIAGNOSIS
The rapid molecular test for Mycobacterium tuberculosis detection is a molecular examination using the semi-quantitative Real-Time PCR Assay (RTPCR) principles with the target of the rpoB gene.
This assay processes the rpoB gene material by DNA automatic extraction in single-use bullets.
The assay technique is using repeated-amplification of the target DNA and fluorimetric detection (Kurniawan et , 2.
Van Rie A et al .
examined suspected tuberculosis with negative Acid-fast bacilli (AFB).
While the sensitivity and specificity of AFB staining were 27% and 99%, the RT-PCR method revealed a 67% sensitivity and 99% specificity, respectively.
Moreover.
AFB also has an advantage in the faster results .
n 2 hour.
, thus allowing to start the therapy on the same day or the next day (Pavsic et al.
, 2.
Examination with the RT-PCR method has a high enough sensitivity value.
It can be an early detection tool to capture patients suffering from pulmonary Meanwhile, a high specificity value can determine whether a person has pulmonary tuberculosis or not.
The RT-PCR method can be used as an early detection tool and as a determinant of the diagnosis of pulmonary tuberculosis, allowing immediate therapy (Khan et al.
, 2.
There was a study getting one sputum specimen with a positive result of the GeneXpert Mycobacterium tuberculosis (MTB) RT-PCR method, but the result of Lowenstein Jensen culture was MTB negative .
rue It is probably because the GeneXpert RTPCR method can detect DNA of dead MTB bacteria in sputum specimens, causing positive MTB detection In sputum culture, bacteria did not grow maybe because the number of living MTB bacteria in the sputum was less than 50-100 bacteria/ml sputum (He et al, 2.
The United States Food and Drug Administration of America approved the two rapid diagnostic tests in the midle of 1990s, they are the direct MTB amplification using a Gen-Probe and MTB nucleic acid amplification examination in patients with positive smear results.
Then in Of the Science FDA approved the .
Journal Health method to be carried out on patients with negative AFB sputum results (Laraque et al.
, 2.
Indonesia, nucleic acid amplication was started to be routinely performed in 2014.
In a medium-sized study, nucleic acid amplification assays used other than airways specimens, such as pleura, lymph nodes, bone marrow, peritoneum, and synovial fluid for MTB rapid diagnosing.
The sensitivity is quite good, namely 64 - 68%, specificity 97 - 100%, negative predictive value 87.
5 - 99.
and positive predictive value 81.
5 - 100%.
A study shows a lower sensitivity rate of pleural biopsy .
Journal Of Health Science Fariz et al.
Nucleic Acid Amplification Test InA.
material, namely 52.
6% (Laraque et al.
, 2.
After cell death or apoptosis, cfDNA is released into Hughes Ralphs et al .
conducted a study on the the bloodstream.
The concentration of cfDNA within cost-effectiveness of the MTB RT-PCR examination in plasma is correlated positively to the numbers of establishing the diagnosis of tuberculosis.
The study cfDNA detached by the tumor and indirectly concluded that RT-PCR examination of MTB in high proportional to the DNA elimination rate through prevalence regions would be more useful in target renal clearance.
cfDNA assays in brain metastases populations with a higher likelihood of tuberculosis.
patients with have low level sensitivity (Rossi et al.
THE
ROLE
NUCLEIC
ACID-BASED
EXAMINATIONS FOR THE DIAGNOSIS OF LUNG
THE
MALIGNANCY
EXAMINATIONS
Molecular examination of lung cancer has become the ROLE
NUCLEIC
FOR
THE
ACID-BASED
LUNG
MYCOSIS
DIAGNOSIS
Thoracic oncologists today faced other Aspergillosis diagnosis is a challenge because current difficulities regarding how to choose examination methods have poor specificity and sensitivity and materials from tissue or fluid biopsies (Abdayem et , 2.
The term liquid biopsy reflects to the histopathological examination of sterile airway examination of circulating tumor DNA within the specimens are the standards for establishing the plasma .
tDNA).
The liquid biopsy also means include diagnosis (Lass-Florl, 2.
Nucleic acid-based the circulating cell-free RNA .
fRNA), the circulating assays such as PCR targeting fungal DNA showed cell-free DNA .
fDNA), the circulating extracellular better sensitivity and specificity, especially if the tumor proteins, vesicles, platelets, and metabolites culture results were negative (Table .
(Buchheidt et (Liu et al.
, 2.
, 2.
Specimen Table 1.
Various of molecular examination and antigen in invasive aspergillosis Methods Galactomannan (GM) Indications Early detection.
Two serum specimens every week, positivity index more than 0,5.
Bronchoalveolar lavages (BAL), positivity .
Journal Of Health Science index above 0,5-1 Lateral device flow assay
Immunochromatographic (LFA) identification using dipstick
PCR
Benefits High risk patient detection.
Adult and child consistent neutropenia > 1 is sign of theraphy failure.
Limitations No non-neutropenia patient data available.
Initial antifungal medication lowered the sensitivity.
Same sensitivity and specificity with GM.
Requiring shorter process in the examination than the GM with serum Rapid early detection.
High sensitivity, and high negative predictive value Not yet available commercially and hard to interpret dipstick assay in several occasions.
Immunocompromise patient More expensive.
Fungi DNA DNA detection.
Specimen extraction technique is hard sample: blood.
BAL, and and collected from complex From: Lass-Florl C.
How to make a fast diagnosis in invasive aspergillosis.
Med Mycol.
57:S155-S160.
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Nucleic Acid Amplification Test InA.
colonization of oropharyngeal bacteria and blood cultures are less sensitive (Murdoch, 2.
Antigen
THE
ROLE
NUCLEIC
ACID-BASED
EXAMINATIONS FOR VIRUS DIAGNOSIS
tests using urine specimens show high but low sensitivity in children.
Several articles conclude that Viral culture examination is the standard and still important in diagnosing viral lung diseases.
Related to the development of molecular-based technology, this examination shows many advantages compared to culture examination.
The biggest advantage is the shorter inspection time until the results come out.
The doctor can start therapy within two to 24 hours of the examined specimen.
Another advantage is good sensitivity and specificity.
Some essays are very sensitive that they need only less number of tested specimen for nucleic acids.
Some viruses, such as PCR testing is not recommended hence the high falsepositive rate (Mothershed, et al.
, 2.
Mycoplasma pneumoniae bacteria are difficult to culture and take a long time.
Therefore, finding these bacteria is often based on serological tests and IgM antibodies with better sensitivity and specificity than serological tests.
Study suggests a positive association PCR Nasopharyngeal, oropharynx, and lower airway specimens show good sensitivity (Mothershed, et al.
Journal Of Health Science Influenza and Respiratory Syncytial Virus, can be detected after clinical signs and symptoms improve Legionella bacteria can infect the lower respiratory (Beck et al.
, 2.
tract and cause community pneumonia.
Several studies on the role of PCR in establishing a diagnosis Ussualy there are several main phases to the inspection process.
The first step is extraction of nucleic acid to obtained genetic molecules from the host cells and the microorganism cells.
The second step is amplification to amplify some parts of the pathogen genome.
The third step is to detect the sensitivity than sputum culture.
But the difficulty is that most sufferers experience dry cough symptoms.
Examination of urine specimens can be a supporting examination of urine antigens (Murdoch, 2.
genetic material that has been amplified on the Chlamydia pneumoniae can be cultured in special cartridge to determine a positive or negative result media but requires a long time and low sensitivity.
(Zhang et al.
, 2.
Most of the findings of these bacteria depend on THE ROLE
NUCLEIC
ACID-BASED
EXAMINATIONS FOR BACTERIAL DIAGNOSIS
Streptococcus pneumoniae is higly prevalent etiology of community acquired lung parenchymal infection in children and adults.
However, definitive level of diagnosis is a challenge because the gold-standard Serological requires the convalescent and acute and serum This explain why the diagnosis can only be established retrospectively.
PCR examination showed lower sensitivity than specimen culture and the specificity value was hard to determine because there .
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Nucleic Acid Amplification Test InA.
standard standards for (Murdoch, 2.
Table 2.
PCR assay in bacteria causing pneumonia Pathogens Streptococcus pneumoniae Gene Targets Autolysin, pneumolysin Specimen Samples Nasopharynx, sputum, urine, serum, plasma, white blood cells.
TNA Results Sensitivity is varied at blood specimen Mycoplasma pneumoniae ATPase operon.
P1 adhesion, 16S rRNA Sputum, nasopharynx.
TNA.
BAL Legionella 16S rRNA, 5S rRNA, mip Chlamydia pneumoniae MOMP, fragmen clone Pst, 16S-23S spacer rRNA, 16S rRNA, 60 kDA protein, 53 kDA protein.
MOMP, 16S rRNA, gseA Serum, sputum.
ETA.
BAL, nasopharynx, serum, white blood cells, urine Nasopharynx.
BAL, oropharyngeal swab, oropharynx, sputum Sensitivity is better than the specimen culture, nasopharynx is more preferred specimen Same sensitivity as the lower respiratory track specimen culture Better sensitivity than the specimen culture Chlamydia psittaci Sputum, nasopharynx.
Not evaluated blood, lung tissue Pneumocystis carinii Mitochondria rRNA, 5S Lung tissue, sputum.
Useful for P.
rRNA, 18S rRNA, nasopharynx.
BAL, pneumonia suspicion with dihyrofolate reductase, oropharynx.
ETA, blood, negative cytology thmidylate sintase.
MSG examination result From: Murdoch DR.
Nucleic acid amplification tests for the diagnosis of pneumonia.
Clin Infect Dis.
:116270.
The Pneumocystis CONCLUSION pneumonia (PCP) diagnosis is staining of cells in bronchoalveolar lavage (BAL) specimens or sputum specimens by immunofluorescence staining, such as Toluidine blue.
Methenamine silver, or Giemsa (Duan et al.
, 2.
Some studies showed that PCR provided better sensitivity and specificity than cytology.
Establishing a diagnosis is not a problem like other causes of pneumonia because the timing of both PCR (Chotiprasitsakul et al.
, 2.
PCR may be preferable to testing sputum specimens in individuales with a significant clinical risk of a history of Human Immunodeficiency Virus (HIV).
PCR assay in bacterial pneumonia are described in Table 2.
Journal Of Health Science Nucleic acid-based testing may be an option for specific diseases and infections.
It plays a significant role in lung cancer.
This examination helps provide the best treatment options that enhance the quality improvement and survival rates.
Nucleic acid assays in pulmonary infectious diseases benefit in accuracy and time required to obtain results.
It is advantageous for the patient to receive definitive therapy.
However, it also has several limitations.
Nucleic acid-based testing is classified as a high-cost assay since advanced technology is involved.
Also, using the essay as a treatment evaluation is not recommended.
Fariz et al.
Nucleic Acid Amplification Test InA.
Conflict of interest declaration The authors have no conflict of interest.
REFERENCES