Epidemiological characteristics of respiratory syncytial virus in hospitalized patients in Guangzhou from 2018 to 2022

Objective To analyze the epidemiological characteristics of respiratory syncytial virus（RSV） before and after the COVID-19 epidemic in Guangzhou in recent years. Methods Nasopharynx swabs from hospitalized patients with acute respiratory infection were collected from two sentinel hospitals in Guangzhou（Guangdong Maternal and Child Health Hospital and Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University） from 2018 to 2022. Luminex respiratory multi-pathogen detection technology was used to detect and type RSV in samples. Results A total of 1 243 nasopharyngeal swab samples were collected. The overall positive rate of RSV was 6. 11%（76/1 243）, including 39 RSV-A（51. 32%, 39/76） and 37 RSV-B（48. 68%, 37/76）. The highest detection rate of RSV in children under 3 years old was 8. 79%（66 cases）. Compared with 2018（8. 30%, 22/265） and 2020（14. 78%, 30/203）, the positive rate of RSV decreased significantly in 2019（3. 13%, 10/319）, 2021（4. 08%, 10/245） and 2022（1. 90%, 4/211）. Compared with 2018（8. 30%,22/265） and 2020（14. 78%,30/203）,the positive rate of RSV decreased significantly in 2019（3. 13%,10/319）, 2021（4. 08%,10/245） and 2022（1. 90%,4/211）. Type A prevailed in 2018（19/22） and 2022（4/4）, type B（25/30） prevailed in 2020, type A and type B coexisted in 2019 and 2021. The detection rate showed had no statistically significant seasonal difference except for 2020 [7. 14%（3/42）, 16. 39%（10/61）, 27. 12%（16/59）, 0（0/42）,χ~2= 16. 975,P＜0. 001]. Among all the 76 RSV positive samples, 17（22. 37%） showed multiple infections. Among them, human rhinovirus was the most common virus causing mixed infection, accounting for 58. 83%（10/17） of the mixed infection. Conclusion RSV is a common respiratory virus prevalent in Guangzhou, and children under 3 years old are the main population infected with RSV. RSV infection is prevalent every other year, with the characteristics of alternating epidemic of type A and type B, and the anti-seasonal epidemic appeared after the COVID-19. After the outbreak of COVID-19, the detection rate of RSV increased significantly in 2020. With the change of the national COVID-19 epidemic prevention and control policy, the detection rate of RSV declined significantly during 2021-2022.

Establishment and application of a triplex RT-PCR method for detection of PDCoV, SADS-CoV, and SVA

[Background] Porcine delta coronavirus（PDCoV）, swine acute diarrhea syndrome coronavirus（SADS-CoV）, and Seneca virus A（SVA） are new pathogens which seriously endanger the development of pig industry. The clinical symptoms of pigs infected with the three pathogens are difficult to distinguish. Therefore, it is urgent to establish a multiplex RT-PCR detection method for rapid diagnosis of suspected pigs and reduce economic losses. [Objective] To establish a triplex RT-PCR method for simultaneous detection of single or mixed infection of PDCoV, SADS-CoV, and SVA. [Methods] Three pairs of specific primers were designed according to the conserved regions of the N genes of PDCoV and SADS-CoV and the L/P1 genes of SVA registered in GenBank, and the optimal annealing temperature（Tm） was determined by temperature gradient PCR method. The primer concentration was optimized by array method. The recombinant plasmids PMD-PDCoV, PMD-SADS-CoV,and PMD-SVA were constructed as standards to determine the limits of detection（LOD）. The specificity of the triplex RT-PCR method was determined with the nucleic acid samples of 6 common pig viruses including porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine reproductive and respiratory syndrome virus. The repeatability of the established method was verified by inter-batch and intra-batch tests. Finally, we employed the triplex RT-PCR method to detect the clinical samples suspected of infection and compared the results with those obtained with the reported detection methods, thus evaluating the clinical application performance of the method. [Results] The optimal Tm was 58.3 ℃, and the optimal primer concentrations were 0.5 μmol/L, 0.25 μmol/L, and 0.25 μmol/L,respectively. The established method had high sensitivity, with the LODs of 1 copy/μL, 1 copy/μL, and 10 copies/μL for PMD-PDCoV, PMD-SADS-CoV, and PMD-SVA, respectively. It had strong specificity, with specific bands only for PDCoV, SADS-CoV, and SVA and no bands for other viruses.Moreover, the method had good repeatability as the test results were consistent between and within batches. The positive rates of PDCoV, SADS-CoV, and SVA in the clinical samples detected by the established method were 65.85%, 30.49%, and 57.32%, respectively, which were consistent with the results obtained with the reported detection methods. Finally, 5 samples were randomly selected from 13 positive samples of PDCoV, SADS-CoV, and SVA for sequencing, and the phylogenetic tree indicated that the PCR amplification sequences of the five positive samples had high homology（above 96%） between each other and also with the reference sequences. [Conclusion] The triplex RT-PCR method established in this study is accurate and reliable for the simultaneous detection of PDCoV,SADS-CoV, and SVA in clinical samples.

Diagnostic Performance and Clinical Utility of Conventional PCR Assay in Early Diagnosis of COVID-19 Associated Rhino-Orbito-Cerebral Mucormycosis.

Early diagnosis and treatment of rhino-orbital-cerebral mucormycosis (ROCM) are crucial. Potassium hydroxide with Calcofluorwhite (KOH + CFW) smears can demonstrate the fungal hyphae, but mixed infections caused by both mucorales and non-mucorales pose a diagnostic challenge. Polymerase chain reaction (PCR) can detect mixed infections and differentiate mucorales from non-mucorales. This study aimed to evaluate the utility of a single reaction PCR in the diagnosis of ROCM and the efficacy of nasal biopsy and endonasal swab in the detection of fungus. Sixty-six clinical samples were collected from 33 patients and were subjected to KOH + CFW smear, culture and PCR. PCR was performed using pan-fungal primers targeting the 28S large subunit rRNA gene, and the amplified products were further sequenced to identify the fungi. KOH + CFW smear, culture and PCR detected mucorales in 54.6%, 27.3% and 63.6% patients, respectively. PCR detected mixed infection in 51.5% patients compared to 9.1% by KOH + CFW smear. PCR detected fungus in 90% of nasal biopsies and 77.8% of endonasal swabs. Rhizopus spp. was the most common fungi identified in 43.2% of PCR-positive samples. PCR is effective in detecting mixed infection and in the diagnosis of ROCM. Nasal biopsies had better fungal detection rates than endonasal swabs.

Co-infection of Klebsiella pneumonia, Cytomegalovirus, Aspergillus and Zygomycete in a patient with SARS-CoV-2.

Co-infection between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other pathogens has become a serious threat. There are the reports of fungal, bacterial, and viral co-infections with SARS-CoV-2. We report the unusual case of concomitant aspergillosis, mucormycosis, cytomegalovirus pneumonia, and also klebsiella pneumoniae empyema as the complication of SARS-CoV-2.

Viral coinfections in COVID-19.

The most consequential challenge raised by coinfection is perhaps the inappropriate generation of recombinant viruses through the exchange of genetic material among different strains. These genetically similar viruses can interfere with the replication process of each other and even compete for the metabolites required for the maintenance of the replication cycle. Due to the similarity in clinical symptoms of most viral respiratory tract infections, and their coincidence with COVID-19, caused by SARS-CoV-2, it is recommended to develop a comprehensive diagnostic panel for detection of respiratory and nonrespiratory viruses through the evaluation of patient samples. Given the resulting changes in blood markers, such as coagulation factors and white blood cell count following virus infection, these markers can be of diagnostic value in the detection of mixed infection in individuals already diagnosed with a certain viral illness. In this review, we seek to investigate the coinfection of SARS-CoV-2 with other respiratory and nonrespiratory viruses to provide novel insights into the development of highly sensitive diagnostics and effective treatment modalities.

Clinical and diagnostic manifestations of tickborne mixed infection in combination with COVID-19.

The coexistence of various pathogens inside the patient's body is one of the poorly studied and current issues. The aim of the study is to identify the relationship between the indicators of complex laboratory diagnostics and the clinical manifestations of a mixed disease during subsequent infection with the SARS-CoV-2 virus using the example of a case of chronic encephalitis-borreliosis infection. Seven blood serum samples were collected from the patient over the course of a year. For the etiological verification of the causative agents of TBE, Lyme disease and COVID-19, the methods of ELISA and PCR diagnostics were used. The patient was diagnosed with Lyme disease on the basis of the detection of IgG antibodies to Borrelia 5 months after the onset of the disease, since she denied the tick bite. In the clinical picture, there was an articular syndrome and erythema migrans. Later, IgG antibodies to the TBEV were found in the blood. Throughout the study, IgM antibodies to Borrelia were not detected. The exacerbation of Lyme disease could be judged by the clinical manifestations of this disease and by the growth of specific IgG antibodies. A feature of this case was that during an exacerbation of the Lyme disease, an infection with the SARS-CoV-2 virus occurred. Treatment (umifenovir, hydroxychloroquine, azithromycin, ceftriaxone) was prescribed, which improved the condition of the underlying disease, decreased joint pain, decreased IgG levels to borrelia. However, during this period, serological markers of TBEV appear: antigen, IgM antibodies, and the titer of IgG antibodies increases. Most likely, this was facilitated by the switching of the immune system to the SARS-CoV-2 virus, with the simultaneous suppression of borrelia with antibiotics and the appointment of hydroxychloroquine, which has an immunosuppressive effect. Despite the activation of the virus, clinical manifestations of TBE were not observed in the patient, which is most likely associated with infection with a weakly virulent TBEV strain. The further course of tick-borne infections revealed the dominant influence of B. burgdorferi in relation to TBEV. Laboratory studies have shown that suppression of the activity of the borreliosis process by etiotropic treatment subsequently led to the activation of the persistent TBEV.

A case of endogenous candida endophthalmitis with incidental cytomegalovirus infection and optic neuropathy in a patient recovered from severe COVID-19.

A 62-year-old female diabetic recovered from COVID-19 pneumonia after receiving a prolonged course of steroids. She presented with a clinical picture of left-eye panuveitis with white cotton ball chorioretinal lesions and RAPD suggesting an optic neuropathy (VA HM). Diagnostic vitrectomy was performed to take samples for infective screen and to give intravitreal voriconazole empirically. Smear, culture, and PCR for viral DNA confirmed mixed infection of endogenous Candida endophthalmitis and incidental CMV infection. With further treatment, her corrected vision improved to 6/18 with regressing fungal lesions in serial fundus photographs. Prompt diagnosis and intervention preserved her vision and prevented potential life-threatening complications.

Potential of Bacteriophage Therapy: A Double Edge Sword to Combat COVID-19 and Associated Pulmonary Bacterial Infections

The Coronavirus Disease 2019 pandemic has wreaked havoc on global health infrastructure and personnel, resulting in enormous misery, deaths and economic stagnation. Severe Acute Respiratory Syndrome-Coronavirus-2 respiratory infections are frequently worsened by secondary bacterial infections and co-infections due to prolonged hospitalizations;resulting in irreversible lung damage, respiratory failure, cardiac arrest and death. The high mortality rate of Coronavirus Disease 2019 patients is primarily due to multi drug resistant microbial (viral/bacterial) infections, unrestrained inflammatory response and delayed antibody production. The superfluous use of broad spectrum antimicrobial drugs as the last resort has further aggravated the Coronavirus Disease 2019 crisis by contributing to the global antimicrobial resistance. To overcome these hurdles for effective treatment of Coronavirus Disease 2019 and associated bacterial infections, phage therapy seems to be promising due to a lack of effective antiviral drugs and antimicrobial-resistant superadded bacterial infections. Prior studies suggest that when phages, their cocktails and endolysins are administered alone or in synergism with antibiotics through nebulization or through intravenous and intraperitoneal injections have exhibited greater antibacterial potential to combat even Multidrug-Resistant pulmonary bacterial infections. Bacteriophages and phagicin have also shown potent antiviral activity by triggering the production of antiviral cytokines. Many studies have also indicated phage mediated antiviral immunity by lowering Nuclear Factor Kappa B activation and reactive oxygen species production. Phage display technique can serve as a promising approach for Coronavirus Disease 2019 vaccine development through production of Severe Acute Respiratory Syndrome-Coronavirus-2 specific antibodies. This review illustrates the potential of phage therapy as a double edged sword to combat both Coronavirus Disease 2019 as well as associated bacterial infections.

Serological cross-reaction and coinfection of dengue and COVID-19 in Asia: Experience from Indonesia.

Similar symptoms and laboratory findings between dengue and coronavirus disease 2019 (COVID-19) pose a diagnostic challenge in some dengue-endemic countries in Asia. In this study, we reported three cases of suspected COVID-19-dengue coinfection in hospitals of Bali, Indonesia. Serological data demonstrated that patients with positive results for dengue virus (DENV) NS1 antigen and anti-dengue IgM were also reactive to COVID-19 rapid antibody tests, suggesting dengue-COVID-19 coinfection. However, two patients were later confirmed negative for SARS-COV-2 by qRT-PCR, implying a plausible cross-reactivity of anti-dengue and anti-COVID-19 antibodies in the serological test. Coinfection of dengue and COVID-19 was evident in one patient, following confirmation of SARS-COV-2 by qRT-PCR and DENV infection using the NS1 antigen serology test. This case was the first case of dengue and COVID-19 coinfection in Indonesia and revealed possible cross-reactivity between SARS-COV-2 and DENV antibodies based on rapid serological tests. Our study indicates a public health concern regarding COVID-19 and dengue detection in Indonesia as well as in other dengue-endemic countries, and it is important for these nations to manage both pathogens concurrently.

[Analysis of adenovirus infection in acute respiratory tract infection cases in Shanghai from 2015 to 2019].

Objective: To study the epidemiological characteristics and mixed infection of adenovirus in acute respiratory tract infections in Shanghai from 2015 to 2019, and to provide scientific basis for the prevention and control of adenovirus. Methods: Acute respiratory tract infections were collected from 3 hospitals in Shanghai from 2015 to 2019. Relevant information was registered and respiratory specimens were sampled for detection of respiratory pathogens by multiplex PCR. Results: A total of 1 543 cases of acute respiratory tract infection were included. The positive rate of adenovirus was 2.92%(45/1 543), the positive rates of influenza like illness (ILI) and severe acute respiratory illness (SARI) were 2.74%(29/1 058) and 3.30%(16/485), respectively. The positive rate of ILI during January-May 2019 was 5.43%(7/129), higher than that in the same period of 2015- 2018 (0.52%-4.48%) (Fisher's exact test value=8.92, P=0.036). The incidence of adenovirus-positive cases was mainly distributed in the first and second quarters, accounting for 62.22% (28/45). The difference of the incidence of adenovirus-positive cases in each quarter was significant (χ(2)= 12.52, P=0.006). The positive rate in the second quarter was highest (6.03%), which was higher than that in other quarters (1.89%-2.93%). There were significant differences among different age groups (χ(2)=16.94, P=0.001), and the positive rate decreased with age (χ(2)=10.16, P=0.001). The positive rate of 13-19 years old group (9.43%) was higher than that of other age groups (1.48%-4.81%). The positive rate of student group (12.07%) was higher than that of other occupations (2.61%). The difference was systematic (χ(2)=11.53, P=0.001). Mixed infection accounted for 31.11% (14/45) of 45 adenovirus positive cases. The mixed infection rates of ILI and SARI were 34.48% (10/29) and 25.00% (4/16), respectively. Among 14 cases of mixed infection, the main mixed infection pathogens of adenovirus were influenza A virus and coronavirus. Conclusion: Adenovirus surveillance should be further strengthened in adolescents with a focus on students and other key groups in the second quarter.