Repiratory infection

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Tuberculosis

Tuberculosis, MTB, or TB (short for tubercle bacillus) is a widespread, and in many cases fatal, infectious disease caused by various strains of mycobacteria, usually Mycobacterium tuberculosis. Tuberculosis typically attacks the lungs, but can also affect other parts of the body. It is spread through the air when people who have an active TB infection cough, sneeze, or otherwise transmit respiratory fluids through the air. Most infections do not have symptoms, known as latent tuberculosis. About one in ten latent infections eventually progresses to active disease which, if left untreated, kills more than 50% of those so infected.

Today, in less-developed countries where population is dense and hygienic standards poor, tuberculosis remains a major fatal disease. The prevalence of the disease has increased in association with the HIV/AIDS epidemic; an estimated one out of every four deaths from tuberculosis involves an individual coinfected with HIV. In addition, the successful elimination of tuberculosis as a major threat to public health in the world has been complicated by the rise of new strains of the tubercle bacillus that are resistant to conventional antibiotics. Infections with these strains are often difficult to treat and require the use of combination drug therapies, sometimes involving the use of five different agents.

One-third of the world's population is thought to have been infected with M. tuberculosis, with new infections occurring in about 1% of the population each year. In 2007, an estimated 13.7 million chronic cases were active globally, while in 2010, an estimated 8.8 million new cases and 1.5 million associated deaths occurred, mostly in developing countries. The absolute number of tuberculosis cases has been decreasing since 2006, and new cases have decreased since 2002. The rate of tuberculosis in different areas varies across the globe; about 80% of the population in many Asian and African countries tests positive in tuberculin tests, while only 5–10% of the United States population tests positive.

The significance of fluorescent quantitativeTB-DNA detection of PCR 

1. Smear staining is the most simple, fast, and economical diagnostic method to directly observe acid-fast bacillus, but the sensitivity of this method is very poor. For Mycobacterium tuberculosis infection, the detection rate is only about 30%. More than 60% of pulmonary tuberculosis and 75% of extrapulmonary tuberculosis could not be detected by the traditional method. The limitation of acid-fast staining is about specificity. It can only  identify Mycobacterium, but cannot differentiate Mycobacterium tuberculosis from other mycobacterium.Positive smear only indicates the presence of acid-fast bacilli, including about 85% of Mycobacterium tuberculosis and 15% of non tuberculosis mycobacteria.

2. Cell culture method for TB diagnosis is generally required to take 4 - 8 weeks to get negative / positive results. It is time-consuming, diagnosis-delaying, and counters to rapid diagnosis of Mycobacterium tuberculosis. 

3. Tuberculin test as auxiliary diagnosis always has no good persuasive, because non tuberculosis mycobacteria have sensitization and cause a false positive. There has not found a specific and pathogenic single antigen or specific and protective single antigen of Mycobacterium tuberculosis. People have made failure to develop a serological kit for diagnosis of tuberculosis in the clinical application. Mycobacterium tuberculosis have a large number of the same antigen protein with some pathogenic or no pathogenic microorganisms, so it is very difficult to find a suitable antigen for ELISA in clinic.

4. Quantitative detection of PCR is a very promising method for rapid diagnosis of Mycobacterium tuberculosis infection in clinical specimens, especially it has practical value for a small amount of bacteria or the specimens that are not easy to be cultured and isolated. At present, Quantitative detection of PCR is approved by State Drug Administration (sFDA) that detects Mycobacterium tuberculosis.

5. Quantitative detection of PCR can stably detect 10 copies of  genomic DNA of Mycobacterium tuberculosis, so it is high sensitivity, strong specificity. A certain proportion of patients were diagnosed as negative by smear or cell culture, but the detection of PCR is positive. the positive rate of Mycobacterium tuberculosis detected by quantitative PCR is significantly higher than acid-fast smear staining of sputum and L-G culture method.

6. If smear staining is positive, and PCR test is negative, the primary reason for consideration is the infection of non Mycobacterium tuberculosis . The next reason we consider is the operation. During the treatment of sputum specimens , if sputum is not enough liquefaction, the bacterium can not be precipitated to the bottom of the tubes during centrifugation and removed with the supernatant. Resulting in false negative results of PCR.

7. Fluorescent quantitative PCR can reflect the changes in the number of Mycobacterium tuberculosis in sputum specimens during the treatment of tuberculosis. It has a certain monitoring effect of anti-tuberculosis drugs. But PCR can not distinguish between viable and dead bacteria, so someone has been cured of tuberculosis, and there is dead Mycobacterium tuberculosis intrapulmonary, so PCR detection is still positive.

8. TB-DNA of infected specimens are amplified and detected at the different sites. It can diagnose whether Mycobacterium tuberculosis exist infection at corresponding part.

① TB-DNA detection in sputum or bronchoalveolar fluid can assist in the diagnosis of pulmonary tuberculosis.
② TB-DNA (white blood cell) detection can assist in the diagnosis of disseminated tuberculosis.
③ TB-DNA detection in CSF can assist in the diagnosis of tuberculosis of central nervous system .
④ TB-DNA detection in cervical swab or urine can assist in the diagnosis of genitourinary tuberculosis.
⑤ TB-DNA detection in pleural and ascitic fluid can assist in the diagnosis of tuberculous pleurisy.

Chlamydia Pneumonia (CP)

Chlamydia pneumoniae was initially recognized as a cause of acute lower respiratory tract infections such as pneumonia and bronchitis in both adults and children, hence the species name "pneumoniae". Moreover, C. pneumoniae has been noted in some individuals to cause a persistent respiratory tract infection following an acute infection, which is entirely consistent with the known chronic nature of all chlamydial infections.

The establishment of such a persistent low-grade infection in the lung by C. pneumoniae creates an important factor for the pathogenesis of this microorganism. The ability of C. pneumoniae to infect a wide variety of human cells including epithelial, endothelial, and smooth muscle cells as well as macrophages, monocytes, and lymphocytes has been well documented. The infection of macrophages, in particular, allows C. pneumoniae to enter into the circulation from pulmonary tissues and can thus cause systemic dissemination. The tendency for C. pneumoniae to disseminate from the initial site of infection in the lung has been described in the murine model of infection. Similar dissemination is presumed to occur in humans. Indeed, the presence of C. pneumoniae DNA in peripheral blood mononuclear cells (PBMCs) has been well documented. In addition, the viability of C. pneumoniae in circulating PBMCs has recently been established.

The ability of C. pneumoniae to cause persistent lung infections combined with its ability to disseminate via the vascular system has raised questions as to the role of this pathogen in a number of chronic diseases including infection-associated chronic fatigue syndrome.

It is not surprising that C. pneumoniae has been reported as a possible cause of infection-associated chronic fatigue syndrome. It is clear that any chronic infection would result in patients experiencing chronic fatigue, thus a chronic chlamydial infection would be expected to do the same. Fibromyalgia and other myalgia of unknown cause have been described in patients with chronic fatigue syndrome; C. pneumoniae antibodies have been linked with myalgia of unknown cause including fibromyalgia.

The significance of fluorescent quantitative CP-DNA detection of PCR  

Chlamydia pneumoniae is strictly intracellular parasite which must grow inside the living cell, so culture condition is greatly strict. generally using the HEp-2t and Hela cell lines to culture it, then using the fluorescent and specific monoclonal antibodies to identify Chlamydia pneumonia. But this method has long cycle and low detection rate, so it is not suitable for clinical detection.

On the serologic methods, micro immunofluorescence (MIF) is sensitive to detect Chlamydia pneumoniae. the specificity of one unit IgM antibody is more than or equal to 1:16 or IgG antibody is more than or equal to 1:512, so it has a certain diagnostic value.

During molecular biological detection, using restriction endonuclease Pst I to digest the DNA of Chlamydia pneumoniae, and can obtain a 474bp DNA fragment, and Chlamydia trachomatis or Chlamydia psittaci do not have the DNA fragment. In fact, the number of pathogens in the specimens are few, so enzyme digestion is often conducted after cell culture or PCR amplification. The enzyme digestion is not suitable for clinical detection because of high cost and complex operation.

Fluorescent quantitative PCR directly amplifys specific nucleic acid fragments of Chlamydia pneumoniae in the sputum, nasal lavage, or opharyngeal swabs or bronchoalveolar fluid. It is simple operation, high sensitivity, strong specificity, and avoid the false positive problem because of conventional complex operation and the pollution of PCR product. It is quickly applied to Chlamydia pneumoniae infection on clinical detection .

The fluorescent quantitative PCR can also be used to evaluate the therapeutic effect for Chlamydia pneumoniae infection. If it is effective treatment, the number of CP-DNA copy in the samples will correspond to drop or be negative.

Mycoplasma Pneumonia (MP)

Mycoplasma pneumoniae (M. pneumoniae) is an "atypical" bacterium (the singular form of bacteria) that causes lung infection. It is a common cause of community-acquired pneumonia (lung infections developed outside of a hospital). M. pneumoniae infections are sometimes referred to as "walking pneumonia." Some experts estimate that between 1 and 10 out of every 50 cases of community-acquired pneumonia in the United States is caused by M. pneumoniae. However, not everyone who is exposed to M. pneumoniae develops pneumonia.

In general, M. pneumoniae infection is a mild illness that is most common in young adults and school-aged children. The most common type of illness caused by these bacteria, especially in children, is tracheobronchitis, commonly called a chest cold. This illness is often seen with other upper respiratory tract symptoms, like a sore throat. Sometimes M. pneumoniae infection can cause pneumonia, a more serious infection of the lungs, which may require treatment or care in a hospital.

Outbreaks of M. pneumoniae occur mostly in crowded environments, like schools, college dormitories, and nursing homes, where transmission is possible through airborne droplets from close person-to-person contact. M. pneumoniae infections also frequently spread within households.

The significance of fluorescent quantitative MP-DNA detection of PCR 

Mycoplasma pneumonia can be cultured from sputum or throat swabs, but culture condition is stringent. The time of isolation and identification requires 1 ~ 2W. Many hospital laboratories can not do the examination. The cycle is too long, and the positive rate is low, so it is a little help for clinical.

The general staining is not easy to dye. Because of no cell wall, there have great plasticity,showing a high degree of polymorphism, so the morphological method difficultly identify it.

Cold agglutination test, only about 50% of patients show positive results, and this reaction is non-specific. Respiratory syncytial virus, influenza, and mumps also can appear the same character with Mycoplasma pneumonia, so it can only serve as auxiliary diagnosis.

Enzyme linked immunosorbent assay (ELISA), immunofluorescence, and immunogold dot method are used to detect the level of serological antibody. IgG (+) can not distinguish between past and present infection. IgM (+) is the sign of emergence of recent acute infection, so it has certain value in diagnosis. It has been reported that the positive rate of IgM is no more than 30%, IgM (-) does not mean excluding MP infection.

It has been reported ELISA method detects the 42kDa of polypeptide or 190kDa P1 of protein antigen of mycoplasma pneumonia in patients with sputum, nasal washings and bronchoalveolar fluid. In fact, the content of the pathogen is very little amount in these specimens, so it is not easily detected. So that the application of the method is restricted for direct detection of protein antigens.

Fluorescent quantitativePCR directly amplifies nucleic acid of pneumonia mycoplasma in sputum, nasal lavage,oropharyngeal swabs or bronchoalveolar fluid. It has the characteristics of rapid, highly sensitive, and strong specificity. It is a broad prospect for the detection of Mycoplasma.

Fluorescent quantitative PCR can also be used to observe the therapeutic effect of Mycoplasma pneumoniae infection. According to the report, After 2 ~ 3W for effective treatment, PCR detection can be all negative.