M.pneumoniae is a bacterial pathogen without a cell wall of the respiratory tract that infects the respiratory tract. The bacterium is also one of the leading causes of acquired pneumonia in the community. The most common manifestations of m.pneumonia include flu-like symptoms such as sore throat, fever, cough, headache, chills, colds, muscle aches, and general discomfort. M.pneumoniae infection occurs worldwide and is most common in children and adolescents. M.pneumoniae is transmitted primarily from close contact with another person. In recent years, pneumonia caused by mycoplasma has increased. Pneumonia caused by M.pneumoniae occurs through damage to the airways of the lungs in a contagious manner and inflammation caused by M.pneumoniae.
The pneumonia caused by this bacteria is generally resistant to treatment with beta-lactam antibiotics. It depends on the structure of the bacterium because it is unable to produce peptidoglycan. However, allergies to other antimicrobial agents are seen in this bacterium. However, resistance to antibiotics in this bacterium has been discussed in many societies. If left untreated, some patients may develop the disease [23].
Data from more than 21 countries showed that m.pneumoniae is the most common type of bacterium that causes atypical pneumonia, which is responsible for creating about 12% of the acquired pneumonia in the community. Past studies on m.pneumoniae have shown that they are 40% more likely to develop acquired pneumonia.. A recent cross-sectional study of people with atopic pneumonia showed generally lower data rates than in any other country. Climate differences, seasons, and improved environmental and public health are likely to reduce the risk of contracting the bacterium. In general, because it is difficult to diagnose mycoplasma biochemistry, little research is available on other bacteria. The slow growth and susceptibility of the bacterium can also double the accuracy and sensitivity of the techniques used to diagnose it.
The main strategy of this study, according to its title, was first to answer the question of what is the prevalence of m.pneumoniae in people with atopic pneumonia. The next step was to measure the drug's resistance to ciprofloxacin in the resulting isolates. The first goal (frequency determination) was performed in three different ways. Cultivation method, PCR molecular method and ELISA method.
In the present study, we joined orthogonal experiment and single-factor tests to optimize several crucial factors in PCR assay based on both the 16S rRNA gene and P1 adhesin gene designed for M. pneumoniae detection.
Gene targets used widely in various types of PCR assays for M. pneumoniae include the P1 adhesin gene and 16S rRNA gene[13]. The P1 adhesin gene is an attractive target for PCR because it repeats up to 10 times within the M. pneumoniae genome, which increases the sensitivity of PCR assay[16]. Another important target is 16S rRNA, or rather rDNA. The advantage of using rDNA sequences is the high degree of conservation of the target and the presence of the highly variable regions[14] .Therefore, it would be greatly valuable for guiding treatment decisions and follow-ups, particularly in countries with a high frequency of strains resistant to antibiotics, because the confirmation of 16S rDNA could be applied in discriminating between bacterial and viral causes of pneumonia.
To our knowledge, however, it is still controversial which target is more effective. For instance, Loens et al. suggested that the P1 adhesin gene may be more sensitive than the 16S rRNA one[13]. Two independent researchers, nevertheless, showed that the amplification of the 16S rRNA gene was more sensitive for the detection of M. pneumoniae because more positive samples were found by 16S rDNA PCR than by a PCR with the P1 gene[14, 15]. The main reason for the ambivalent conclusions is that the researchers detected the DNA directly from clinical samples, rather than a standard strain DNA of M. pneumonia, to compare the sensitivity of 16S rDNA PCR with P1 gene PCR.
These results confirmed that the 16S rRNA gene primers are more sensitive than the P1 adhesin gene primers, as the 16S rRNA gene primers can detect up to 10 fg of M. pneumoniae DNA and the P1 gene primers can detect 100 fg of M. pneumoniae DNA at most. This was mainly because the presence of approximately 103 copies of 16S rRNA per mycoplasma cell and the high degree of conservation of the rRNA genes allowed a high fixation of primers on the target and lead to a higher PCR yield[4, 14] .Importantly, because RNA is destroyed more rapidly than DNA after the death of the mycoplasma cell, detection of the 16S rRNA gene provides further evidence of viable mycoplasmas in the specimen [4].
At this stage, part of the 16SrRNA gene of this bacteria was replicated. Propagation was performed using mycoplasma-specific primers. The second step was to use the PCR technique using specific P1 gene primers.
Primer F has a general structure, but primer R (return) has a specific structure for screening for mycoplasma. In 2014, Tabatabai Qomi et al. Used these primers to examine the presence of mycoplasma as a contaminant in cellular categories. In addition to M.pneumoniae, they identified other bacteria of this genus. Bacteria including Mycoplasma arginini, Mycoplasma hyorinis, Mycoplasma orale, Mycoplasma synoviae, Mycoplasma gallinarum were also identified[24] .
We observed that 0.14% (14/100) of the specimens were positive with the 16S rRNA gene PCR and 7% (7/100) of the specimens were positive with the P1 adhesin gene PCR.
The first use of these primers dates back to 1995 and research by Pruckler et al. The aim of this study was to study the species of M.pneumoniae that can be traced in cellular categories [25].
In 2014, Mirihan et al. Used this primer to study the infection with Mycoplasma bacteria in patients with pneumonia admitted to Egyptian hospitals. They assessed a prevalence rate of 22.94% among 170 individuals. In the present study, a prevalence of 14% was observed, which is lower than that of Mirihan et al. The reason for this decrease is probably related to Iran's health conditions [26].
Determination of M.pneumonea species was also determined by specific proliferation of P1 gene. The results of this study showed that 7 samples are infected with M.pneumoniae. Another 7 samples were positively evaluated for mycoplasma. The identity of the infected species remained unknown.
In 2015, Amirian et al. In Saveh city used PCR method to diagnose M.pneumoniae using P1 gene proliferation. Out of 120 people with chronic lung infection, 8 were positive, with the highest frequency in the 21-year-old age group and the lowest in the 3-year-old age group [27].
The positive PCR result is based on the 16SrRNA gene, but the lack of bacterial growth is due to the origin of the microbial agent. The bacteria is present and is made of mycoplasma but is unable to grow. Yoshida et al., Using similar primers and replicating part of 16SrRNA, isolated the specific species of birds related to the genus Mycoplasma [28].
Cultivation is rarely used for diagnosis of M. pneumoniae infection in most clinical laboratories because the fastidious growth requirements and length of time necessary to culture M. pneumoniae (three to six weeks) make growing the organism impractical for patient management[4, 10].
Currently, serological assay is the most widely used means for laboratory confirmation of mycoplasmal respiratory infections [10]. However, there are concerns about the use of single qualitative tests to identify acute M. pneumoniae infections in adults, since many persons may not mount an IgM response, presumably because of re-infection, and when it is produced, IgM may persist for long periods [19, 29, 30]. Furthermore, the percentage of individuals with acute infection who demonstrated a positive IgG response in the acute phase was less than 50% in a recent study.
It has been suggested that cross-reactivity with antigen preparations used in some of the commercial enzyme immunoassays (EIAs) result in over-diagnosis of M.pneumoniae infections[29]. Therefore, even if serology is a useful epidemiologic tool in areas where the infection rate of M. pneumoniae is high, it is less suited for assessment of individual patients in clinical laboratories[11]. Compared with serology and culture, a direct detection of pathogens in clinical specimens has been done more regularly using molecular biology techniques. PCR approaches have been the most valuable method for rapid, sensitive, and specific diagnosis of M. pneumoniae infection[4]. However, the application of molecular methods of enhanced sensitivity may be necessary since the pathogens are probably present in small quantities.
In 2003, Orsi et al. Evaluated the performance of four PCR-based M.pneumoniae identification methods. In this study, false PCR results were confirmed. This study proves that bacterial culture is the most reliable way to measure mycoplasma in terms of false positives. However, based on this study, the false negative results of the cultivation technique are high[13].
In 2006, Shah Hosseini et al., Using culture and PCR methods, compared the performance of these two methods to identify pneumococcal mycoplasma in people with acquired pneumonia. According to their research, all cases of positive culture in terms of PCR will also be positive. Therefore, PCR was considered an accurate and hypersensitive method of culture [31].
In 2013, in a study using serology and PCR, Noorbakhsh etal.Studied the role of m.pneumoniae in children with adenoid hypertrophy and rhinosinusitis. Positive PCR results were reported in 35% of the samples. A survey of antibody titers by ELISA also found that 10 percent of people with IgM and 20 percent had IgG in their serum. In this mycoplasma, pneumonia was also detected using PCR in adenoid tissue samples[32] .
In the present study, data from the ELISA method showed that out of a population of 100 people studied, 19 contained specific anticoagulants of M.pneumoniae. This rate is negligible compared to many other studies. In 2002, for example, Rastawick et al. Found that 63% of the 66 patients admitted to Polish hospitals had specific IgG antibodies specific to m.pneumoniae [23]. This high level is probably dependent on factors such as the antibody titer determination kit.
In addition, reviewing the ELISA results and comparing it to the PCR test reveals a valuable point. All seven samples infected with M.pneumoniae were also positive for ELISA. This shows the importance of positive ELISA results. However, species of the genus Mycoplasma (other than Mycoplasma pneumoniae) were unable to increase its antibody titer.
in 2001, Suni et al. Used the ELISA method to investigate the prevalence of Mycoplasma pneumoniae infection in people with acute and non-acute pneumonia. They showed that 92 out of 210 people tested positive for IgG mycoplasma pneumoniae in their serum [33].
Sharifi et al. In 2011 in Tabriz, using PCR, culture and ELISA methods, examined the frequency of respiratory infections caused by M.pneumoniae. In addition, they measured patients' IgM antibody titers. Out of 200 patients with pneumonia, 12 samples were evaluated by PCR method infected with this bacterium. Also, 4 samples were isolated by m.pneumoniae bacterial culture method. Using ELISA technique, it has been proven that there are 10 mycoplasmal IgM antibodies in the serum of individuals [34].
In 2004, in Tehran and in Tehran, Niakan et al. Used the ELISA method to investigate the serological frequency of IgG associated with m.pneumoniae in people with atypical pneumonia. ELISA method was used in their research. Blood serum was assessed in 104 individuals, of which 57% were positive. 36% were negative and 5% were intermediate [35].
In this study, samples tested positive for M.pneumoniae were evaluated for MIC in terms of ciprofloxacin. The result of this evaluation was resistance 2 separation. Restriction of m.pneumoniae in treatment (intrinsic resistance to beta-lactam antibiotics) has led to the treatment of this bacterium as a sensitive issue. There is an increase. For example, 80 to 90 percent prevalence of macrolide resistance leads to ineffectiveness as one of the most important and effective factors in their treatment [36].
On the other hand, research on m.pneumoniae and its antimicrobial properties is much more difficult than other bacteria. Therefore, the main focus is on the limited scientific reports provided from various sources. This has led to the fact that these reports, like the report from the present study, are of particular importance. The prevalence of 2 resistant isolates among the 8 resistant isolates (in the present study) compared to the prevalence reported in the Gruson study, and colleagues reported the resistance of this bacterium against resistant strains. It had the highest resistance among all antibiotics. The maximum reported MIC for ciprofloxacin in the Gruson study was 128 micrograms per milliliter. And the minimum MIC value was 1 microgram per milliliter. The fact that the bacterium Mycoplasma pneumoniae is more resistant to ciprofloxacin than other antibiotics is a warning sign of an increase in its prevalence. However, in our country, this increase in resistance is not seen. To confirm this, we can refer to the research of Kashmiri et al. In 2014. In general, the prevalence of resistance is 2 isolates less than 7 isolates than the 81% resistance of macrolides in Japan [37]and less than 7.5% of the resistance of levofloxacin in Europe (France) [38].
In 2011, Aizhen et al. Investigated the therapeutic response to concomitant use of ciprofloxacin and corticosteroids in the treatment of infections caused by m.pneumoniae. They found combination therapy to be effective. However, some people experience delayed fever and symptoms that appear to be genetically predisposed. This study shows that the patient's genetics are involved in the symptoms and treatment of pneumococcal mycoplasma [39].
In 2014, Biljana et al. Examined the role of community-acquired pneumonia created by M.pneumoniae. 166 children aged 1 to 15 years were assessed. In this study, serological methods and RT PCR technique were used. Specific IgM and IgG antibodies to m.pneumoniae were measured in patients' serum. During this study, the clinical symptoms of M.pneumoniae, including headache, wheezing, were evaluated. It was also found that 14.5% of people with m.pneumonia [40].