Epidemiological characteristics of 369 tuberculosis patients with multidrug resistance inHainan Island: A cross-sectional study

Xiu-Juan Zhao, Lin Liu, Xing-Yong Wu, Hui-Min Fu, Biao Liu, Hua Pei,Qian-Feng Xia✉

1. Laboratory of Tropical Biomedicine and Biotechnology, Hainan Medical University, Haikou, Haikou571199, China

2. Key Laboratory of Tropical Translalional Medicine of Ministry of Education and School of Tropical Medicine and Laboratory Medicine, Hainan medical University, Haikou571199, China

3. Public Health School, Hainan Medical University, Haikou 571199, China

4. Department of Clinical Laboratory, The Second Affiliated Hospital, Hainan Medical University, Haikou 571199, China

Keywords:Mycobacterium tuberculosis Multi-drug resistant tuberculosis Time distribution Regional distribution Population distribution

ABSTRACT Objective: To investigate the epidemic characteristics of multidrug resistant tuberculosis in Hainan, China, to provide basic information and theories for the prevention and control of multidrug resistant tuberculosis. Methods: A retrospective descriptive epidemiological study was conducted. Data of multidrug resistant tuberculosis in Hainan Island from January 1,2014 to December 31, 2019 were collected for statistical analysis. The counting data were described by frequency (percentage), and the measurement data were described by mean ±standard deviation. Chi square test was used to compare the differences between different groups. If P ＜ 0.05, the difference was statistically significant. Results: From 2014 to 2019,the multidrug resistant rate of tuberculosis in Hainan Island was 24.9% (369 / 1484), 8.0%(63 / 791) in newly-treated tuberculosis, and 44.2% (306 / 693) in retreatedmultidrug resistant rate of tuberculosis in Hainan Island was 24.9% (369 / 1484), 8.0% (63 / 791) in newlytreatedtuberculosis, and 44.2% (306 / 693) in retreated tuberculosis. The multidrug resistant rate of newly-treated tuberculosis decreased by 0.05% per year, while that of retreated tuberculosis increased by 0.03% per year. multidrug resistant tuberculosis was mainly distributed in the city of Haikou (83 cases), Danzhou (44 cases), Wenchang (42 cases),Wanning (37 cases) and Dongfang (32 cases), accounting for 64.5% of all multidrug resistant tuberculosis. Among 369 multidrug resistant tuberculosis, 81.0% were male. The proportion of newly-treated cases was 17.1% (63 / 369), while the proportion of retreated cases was 82.9%(306 / 369), which was significantly higher than that of newly-treated cases. The multidrug resistant rate of tuberculosis in different years was different. Conclusion: The drug resistance rate of tuberculosis in Hainan Island is higher than the national average level, which needs attention. The epidemiological characteristics of multidrug resistant tuberculosis with different treatment history were different.

In the late December 2019, COVID-19, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), began to spread in Wuhan, China. Since the outbreak of COVID-19, many countries and regions around the world have suffered significant harm and challenges. As of 30 November 2021, the number of confirmed COVID-19 cases worldwide has exceeded 260 million,and the cumulative death toll has reached over 5.2 million[1]. Most COVID-19 patients have typical clinical symptoms, but with further research on COVID-19 and improved detection techniques for SARS-CoV-2, the number of asymptomatic COVID-19 patients is increasing, and many asymptomatic COVID-19 patients are easily overlooked, because they have no obvious clinical symptoms,However, due to the presence of SARS-CoV-2 in their bodies,they were able to transmit SARS-CoV-2 to others, which greatly accelerated the worldwide pandemic and brought great difficulty and threat to global epidemic prevention and control. Therefore,early detection and identification of asymptomatic infected persons through various testing methods are effective measures to control the COVID-19 pandemic. This article will provide a brief review of the available methods and techniques for detecting asymptomatic COVID-19 patients.

1. COVID-19

COVID-19 is an emerging respiratory infectious disease caused by SARS-CoV-2. The pathogen SARS-CoV-2 is a β-coronavirus of positive single-stranded RNA virus, which is spherical or oval in shape with protuberant glycoproteins on its surface. Under electron microscope, it looks like a tree crown. It is highly infectious and pathogenic.[2] Studies[3-4] found that SARS-CoV-2 invaded human cells mediated by angiotensin converting enzyme 2 (ACE2), and the binding force between SARS-CoV-2 and ACE2 is about 4 times that of SARS virus, which may also be one of the reasons why COVID-19 is more infectious than SARS.

COVID-19 can be caused by SARS-CoV-2 infection in humans through respiratory droplets, contact, and fecal transmission. The main clinical manifestations of COVID-19 include respiratory symptoms such as cough, expectoration, sore throat and dyspnea;gastrointestinal symptoms such as nausea, vomiting, diarrhea and abdominal discomfort; and systemic symptoms such as fever, fatigue and muscle soreness.

2. Asymptomatic COVID-19 patients

Asymptomatic COVID-19 carriers are those who test positive for SARS-COV-2 etiology in respiratory tract specimens, but do not have self-perceived clinical manifestations, such as fever, dry cough, sore throat, etc. The clinical characteristics of asymptomatic COVID-19 infected persons are as follows[5][7]: (1) positive results of reverse transcription polymerase chain reaction (RT-PCR) of two nasal swabs and(or) pharyngeal swabs within 24 hours;(2) No selfperceived clinical symptoms, such as fever, cough, fatigue, sore throat, muscle pain, etc.;(3) SARS-COV-2 was positive in respiratory specimens with or without the pulmonary imaging changes that could be detected by chest computed tomography(CT).

Asymptomatic COVID-19 patients have no obvious clinical manifestations, but their positive SARS-COV-2 etiology tests in respiratory specimens indicate the presence of SARS-COV-2 in their bodies, so they can cause the spread and spread of the epidemic.

3. Testing methods for asymptomatic COVID-19 infected persons

3.1 Acid test nucleic acid test

Nucleic acid was detected by molecular biology techniques (reverse transcription-polymerase chain reaction, thermostatic expansion, in situ hybridization and sequencing, etc.) for all kinds of body fluids,or tissue specimens were pathogen nucleic acids (DNA or RNA)in the structure, content and subcellular localization of qualitative quantitative analysis, to determine whether specimens of pathogen infection, so as to help clinical diagnosis of disease.[8] Nucleic acid detection for COVID-19 by reverse transcription-polymerase chain reaction (RT-PCR) is not only the officially recognized SARSCoV-2 determination method, but also the main detection method for screening asymptomatic infected persons at present.[9] The detection method is usually divided into two steps, namely nucleic acid extraction and PCR amplification, and the total duration is about 3-3.5 h.[10] Because SARS-CoV-2 can be transmitted early in infection, RT-PCR detection of sarS-CoV-2 is particularly suitable for early screening of asymptomatic infected persons.

Common sample collection methods in nucleic acid testing for COVID-19 include nasal swabs, pharyngeal swabs and anal swabs.However, at present, samples for COVID-19 are mainly collected by nasal swabs and(or) pharyngeal swabs. This is because virus infection generally starts from the upper respiratory tract, develops from the nasopharynx to the pharynx, and finally develops to the lower respiratory tract. Nasopharynx is the first portal for viruses to invade the human body, so the viral load of respiratory tract specimens is high[11]. In addition, the sampling method is simple,quick and easy to operate. Studies[12] have shown that nasal swab samples are more sensitive than pharyngeal swab samples during RTPCR detection. However, nasal swab sampling will cause mucosal damage to tissues of patients, especially for patients who have been in hospital for a long time and need to take multiple samples,resulting in decreased cooperation and reluctance to retain samples,which is not conducive to diagnosis and treatment evaluation of patients[13].In addition, Wu et al.[14] found that the duration of nucleic acid positive in feces or anal swab of some asymptomatic or mild infected persons was longer than the duration of upper respiratory tract. Another study[15] found that the positive rates of nucleic acid in the first to fourth week of exposure of asymptomatic infected persons were 40.72%, 97.64%, 58.72% and 18.42%, respectively,and the positive rates of nucleic acid in feces were 8.97%, 18.05%,51.38% and 27.24%, respectively. Therefore, adding nucleic acid test of anal swab at 3-4 weeks after infection can improve the detection rate of asymptomatic infected persons and reduce missed diagnosis.However, considering the convenience and privacy of specimen collection, nasopharyngeal swabs and anal swabs are only collected from key populations such as isolation sites at the same time.

Due to the insufficient viral load of asymptomatic infected persons, non-standard handling of specimen collection, storage and transportation, poor sensitivity of in vitro diagnostic reagents, and insufficient clinical laboratory testing capacity, the sensitivity of nucleic acid testing will be affected to a large extent, which may lead to missed diagnosis or delayed effective diagnosis. Therefore, for patients with highly suspected SARS-CoV-2, multiple sarS-CoV-2 testing methods should be combined to improve the success rate of nucleic acid testing and reduce the occurrence of false negative and negative repositive results.

3.2 Primary antigen detection

COVID-19 antigen detection refers to the direct detection of pathogens in samples by specific antibodies of SARS-CoV-2.The applicable samples are generally from infected sites or blood,mainly nasopharyngeal swabs, vessel-lavage fluids, serum, etc.[10].The detected antigen types are mainly the spike glycoprotein (S)and nucleocapsid protein (N) of SARS-CoV-2[16], and the detection results can be used as direct evidence for early diagnosis of infection of this pathogen.

Immunochromatography is the most common method for detecting SARS-CoV-2 antigen.[17] Diao et al.[18] Developed a fluorescent immunoassay (FIC) for the specific detection of SARS-CoV-2 N protein antigens. This assay can detect SARS-CoV-2 infection in 10 minutes without expensive equipment. They then used this method to qualitatively detect sars-cov-2n protein antigens in nasopharyngeal swab samples from suspected COVID-19 inpatients or outpatients from eight central hospitals in China. The results showed that when the threshold of sars-cov-2ct was 40copies/mL,The sensitivity, specificity, and accuracy of the N protein antigen assay were 75.6%(95% CI 69.0-81.3), 100%(95% CI 91.1-100), and 80.5%(95% CI 75.1-84.9), respectively. The results show that the N protein antigen detection has high specificity, but its sensitivity is relatively low.

In addition, Lorena et al.[17] used Bioeasy's novel SARSCOV-2 antigen detection kit to detect SARS-CoV-2 antigen in nasopharyngeal and oropharyngeal swabs from 127 suspected cases and investigated its accuracy. The results showed that the overall sensitivity and specificity were 93.9%(95%CI86.5-97.4%) and 100%(95%CI92.1-100%), respectively. Compared with the results of RT-PCR as a reference, the accuracy of the detection method was 96.1%, and the sensitivity was significantly reduced in the subgroup samples with Ct value ＞25.1.Sensitivity was significantly higher in samples with high viral loads.

It can be seen that the sensitivity and specificity of the above two COVID-19 antigen tests are ideal, but the results are greatly affected by sample quality, sampling site and virus expression level. Therefore, this detection method should not replace the role of RT-PCR nucleic acid detection in the diagnosis and screening of SARSCOV-2 infection. How to reduce the false negative rate of COVID-19 antigen test in low viral load samples is a key issue for clinical use of this test.

3.3 Clear body test Serum antibody test

Serum antibody testing for COVID-19 involves taking blood samples from subjects to detect the presence and concentration levels of antibodies against SARS-COV-2 S and N proteins in the serum to determine whether the body is fighting SARS-COV-2. IgM is the first antibody produced when infection occurs in the human body,and IgG is the most common and abundant antibody in serum. It can neutralize the pathogen by binding and inactivating the S and N proteins of SARS-CoV-2.[19] Serum antibody detection for COVID-19 is developing rapidly as an effective supplement to nucleic acid detection. China's COVID-19 Diagnosis and Treatment Protocol(Trial Eighth Edition)[20] explicitly includes antibody detection results in the diagnostic criteria for confirmed COVID-19 infection and the exclusion criteria for suspected cases. There is also much evidence that it plays an important role in diagnosing asymptomatic COVID-19 infections.

Lei et al.[21] randomly collected serum samples from 63 asymptomatic infected patients, and detected IgM and IgG antibodies against SARS-CoV-2 S protein and N protein in these serum samples by chemiluminescence method, and found that the positive rate of nucleic acid test combined with IgM antibody serological test was 55.5%. The positive rate of nucleic acid test combined with IgG antibody serological test was 12.7%, while that of nucleic acid test alone was 6.3%. This indicates that the sensitivity of nucleic acid detection combined with serological antibody detection, especially combined with IgM antibody detection, is significantly higher than that of nucleic acid detection alone.

In addition, Liu et al.[22] evaluated two ELISA kits based on SARS-CoV-2S protein and N protein for detection of IgM and IgG antibodies. The sensitivity of S-protein-based IgG and IgM to the kit was 74.3% and 77.1%, respectively, while the sensitivity of N-protein-based IgM and IgG to the kit was 70.1% and 68.2%,respectively. It can be seen that ELISA based on S protein has higher sensitivity than ELISA based on N protein.

In addition, serological tests are simple and low-cost, making them suitable for mass screening. However, studies[23] show that the accuracy of antibody detection is 30.1% in the first week after infection, 72.2% in the second week, and 91.4% after three weeks.It is clear that serum antibody testing is not appropriate in the early stages of SARS-CoV-2 infection and does not have a significant diagnostic role until 2-3 weeks after infection.

3.4 Chest CT scanning

Chest CT scan is a common imaging examination method for auxiliary diagnosis of respiratory diseases by using CT equipment to perform tomography examination on the chest, mainly in the lungs and mediastinum. Under normal circumstances, there is more air in both lungs, and the density of air on CT image is very low, which is easy to form a good contrast with other tissue structures.The most common CT findings of COVID-19 patients are multiple ground glass shadows in the subpleural and peripulmonary zones,with or without mixed consolidation. Some lesions can be seen around the bronchovascular bundle. The lesions are commonly patchy, triangular or wedge-like, round, and central-lobular nodules with blurred boundaries and accompanied by halo or counter-halo.[24]

Li Gang et al.[25] conducted a retrospective study on chest CT images of 56 asymptomatic COVID-19 infected patients in a wuhan squatted hospital, and found that 7 patients (12.5%) showed no obvious abnormality on chest CT images, while the other 49 patients(87.5%) showed obvious lesions on chest images, which were mostly in both lungs (69.4%).

Chen et al.[26] performed chest CT scans on 20 asymptomatic infected patients who tested positive for COVID-19 nucleic acid (by RT-PCR) or serum antibody. The results showed that 47.62%(95%CI:31.13%-72.87%) of the patients had pulmonary abnormalities, and the most common abnormal manifestation was ground glass opacity 41.11%(95%CI: 19.7%-85.79%). The secondary manifestations were unilateral pneumitis 30.95%(95%CI:22.22%-43.12%) and bilateral pneumonia 27.13%(95%CI:1.7.28%-42.59%).

In conclusion, chest CT examination has a good diagnostic value for asymptomatic COVID-19 patients, and can provide an important basis for clinical treatment decisions and epidemic prevention and control.

3.5 Application of biosensor for physical sensation

Biosensor is a sensor that uses the activity phenomenon of microorganism or living body in biological tissue as the transformation structure. It can make biological active material(enzyme, protein, DNA, RNA, antigen, antibody, etc.) organically combine with physical and chemical transducer, so as to detect the concentration of the object to be measured. Many researchers are working to develop a rapid diagnosis of COVID - 19 biosensor,such as ring mediated isothermal amplification (LAMP), has been widely applied in the detection of multiple pathogens,Including severe Acute respiratory syndrome coronavirus (SARS-CoV) and

Middle East Respiratory syndrome coronavirus (MERS-CoV).[27-28] Electrochemical methods can distinguish small changes from the recognition sites on the electrode surface, and can be used for labeling free detection without single antibody.[29]

Zhu et al.[30] designed a multiple reverse transcription-loopmediated isothermal amplification (mRT-LAMP) and nanoparticle based lateral flow biosensor (LFB) detection method (mRT-LAMPLFB) to diagnose COVID-19. To verify the applicability of mRTLAMP-LFB method for detection of SARS-CoV-2, oral and pharyngeal swab samples from 33 COVID-19 confirmed patients and 96 non-COVID-19 patients in Sanya People's Hospital of Hainan Province were tested using mRT-LAMP-LFB method.Detection of SARS-CoV-2 was 100% sensitive (33/33 oropharyngeal swab samples from COVID-19 patients) and 100% specific (96/96 oropharyngeal swab samples from non-COVID-19 patients). In addition, the method can complete the whole diagnostic process from sample collection to result interpretation within 1 hour.Therefore, RT-LAMP-LFB is a rapid, sensitive and reliable detection technique with high sensitivity and specificity for the diagnosis of SARS-CoV-2 infection, and is expected to become a common diagnostic method for asymptomatic COVID-19 patients. However,with the widespread spread of SARS-CoV-2 virus, the accuracy of MRT-LAMP-LFB technology may be affected by mutations in the target gene primer region.

Abrego-Martinez et al.[31] also proposed an electrochemical biosensor based on aptamers to detect SARS-CoV-2S protein. The sensor can be directly used in a small, low-cost potentiostat coupled to a smartphone, i.e. a handheld potentiostat can be combined with a smartphone to quickly detect sarS-CoV-2 S protein directly on the smartphone screen in real time. However, the sensor has not been proven to be reliable for diagnosing COVID-19 in clinical trials. If the sensitivity and specificity of the sensor for diagnosing COVID-19 and asymptomatic infected persons can be confirmed in clinical trials, it may become a promising COVID-19 detection tool.

4. Summary and prospect

The global ravages of COVID-19 pose a serious threat to the physical and mental health of people in many countries and regions of the world, and the increasing number of asymptomatic COVID-19 infections have largely accelerated this pandemic. Therefore, early detection of asymptomatic infected persons through various testing methods is essential to control the spread of SARS-CoV-2.

Nucleic acid test is currently the main test method for screening asymptomatic COVID-19 infected persons. However, due to insufficient viral load of infected persons, improper handling of specimens and other problems, the sensitivity of nucleic acid test will be affected to a large extent. SARS-CoV-2 antigen detection has high specificity and sensitivity, but the detection results are greatly affected by sample quality, infection site and virus expression.Serological antibody detection method is simple to operate and low cost, and can be used in combination with nucleic acid detection to improve the sensitivity and accuracy of diagnosis. Chest CT scan has good diagnostic value for asymptomatic COVID-19 patients and can provide an important basis for clinical treatment decisions and epidemic prevention and control. Finally, the development of biosensor and other new technologies can promote the diagnosis of COV-ID-19 asymptomatic infection, which is expected to be better verified in clinical trials.

Although most of the methods and techniques reviewed in this paper have good sensitivity and specificity for the detection of asymptomatic COVID-19 infected persons, there are some limitations. In the face of the recurrent COVID-19 epidemic and the rapid spread of SARS-CoV-2 virus, we still urgently need to further explore and improve methods and technologies for detecting asymptomatic COVID-19 patients.

Declaration of conflict of interest

All authors declare that there is no conflict of interest.

The author contributions Jiang Jing-jing: Topic selection and writing; Gao Chun, Feng Fujuan: Literature collection and collation; Yu Xiao-hui: Guide the revision of the first draft; Zhang Jiu-cong: Topic selection and design.