Clinical Evaluation of a Multiplex PCR Assay for Simultaneous Detection of 18 Respiratory Pathogens in Patients with Acute Respiratory Infections.

Reliable diagnostics are necessary to identify influenza infections, and coronavirus disease 2019 (COVID-19) highlights the need to develop highly specific and sensitive viral detection methods to distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory pathogens to prevent their further spread. In this prospective study, 1070 clinical respiratory samples were collected from patients with acute respiratory infections from January 2019 to February 2021 to evaluate the diagnostic performance of a multiplex probe amplification (MPA) assay, designed to screen 18 pathogens, mainly those causing acute respiratory infections. Ninety-six positive samples and twenty negative samples for the 18 respiratory pathogens defined by the MPA assay and reverse transcription polymerase chain reaction (RT-PCR) were further confirmed by reference next-generation sequencing (NGS). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the MPA assay were 95.00%, 93.75%, 98.96% and 75.00%, respectively. Additionally, the co-infection rate for these positive samples were 25% (24/95). The MPA assay demonstrated a highly concordant diagnostic performance with NGS in the diagnosis of 18 respiratory pathogens and might play an important role in clinical respiratory pathogen diagnosis.

Pneumonia Caused by Coinfection with Cytomegalovirus and Pneumocystis jirovecii in an HIV-Negative Infant Diagnosed by Metagenomic Next-Generation Sequencing.

Background: Pneumonia produced by coinfection with Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) in infants and young children without timely diagnosis and treatment is often fatal due to the limitations of traditional tests. More accurate and rapid diagnostic methods for multiple infections are urgently needed. Case Presentation: Here, we report a case of a 2-month-old boy with pneumonia caused by Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) without HIV infection. Chest computed tomography (CT) showed massive exudative consolidation in both lungs. Microscopic examination of stained sputum and smear specimens and bacterial and fungal culture tests were all negative, and CMV nucleic acid and antibody tests were positive. After a period of antiviral and anti-infective therapy, pulmonary inflammation was not relieved. Subsequently, sputum and venous blood samples were analysed by metagenomic next-generation sequencing (mNGS), and the sequences of PJ and CMV were acquired. The patient was finally diagnosed with pneumonia caused by PJ and CMV coinfection. Anti-fungal combined with anti-viral therapy was given immediately. mNGS re-examination of bronchoalveolar lavage fluid (BALF) also revealed the same primary pathogen. Therapy was stopped due to the request of the patient's guardian. Hence, the child was discharged from the hospital and eventually died. Conclusion: This case emphasizes the combined use of mNGS and traditional tests in the clinical diagnosis of mixed lung infections in infants without HIV infection. mNGS is a new adjunctive diagnostic method that can rapidly discriminate multiple causes of pneumonia.

RT-LAMP assay for rapid detection of the R203M mutation in SARS-CoV-2 Delta variant.

The highly infectious Delta variant strain of SARS-CoV-2 remains globally dominant and undermines COVID-19 vaccines. Rapid detection of the Delta variant is crucial for the identification and quarantine of infected individuals. In this study, our aim was to design and validate a genotyping RT-LAMP method to detect Delta variants specifically. R203M in the N gene of SARS-CoV-2 was chosen as the Delta variant-specific mutation for genotyping. To target the R203M-harboring region and the conserved sequence of the N gene, two sets of primers were designed, and a Cq (quantification cycle) ratio-based RT-LAMP for SARS-CoV-2 and R203M detection was developed by analyzing the significant discrepancy in amplification efficiency of the two sets of primers. We validated the RT-LAMP method on 498 clinical specimens in parallel with RT-qPCR, and 84 Delta variants from 198 positive samples were determined by sequencing. Compared with traditional RT-qPCR analyses, RT-LAMP appears to be 100% accurate in detecting SARS-CoV-2 clinical samples. RT-LAMP has a good ability to distinguish between Delta and non-Delta variants under a Cq ratio threshold of 1.80. Furthermore, the AUC (area under the curve) of this method was 1.00; the sensitivity, specificity and accuracy were all 100%. In summary, we have proposed a rapid, accurate and cost-effective RT-LAMP method to detect SARS-CoV-2 and Delta variants, which may facilitate the surveillance of COVID-19.

Epidemiological, Virological and Serological Features of Coronavirus Disease 2019 (COVID-19) Cases in People Living With Human Immunodeficiency Virus in Wuhan: A Population-based Cohort Study.

BACKGROUND: We aimed to describe the epidemiological, virological, and serological features of coronavirus disease 2019 (COVID-19) cases in people living with human immunodeficiency virus (HIV; PLWH). METHODS: This population-based cohort study identified all COVID-19 cases among all PLWH in Wuhan, China, by 16 April 2020. The epidemiological, virological, and serological features were analyzed based on the demographic data, temporal profile of nucleic acid test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the disease, and SARS-CoV-2-specific immunoglobin (Ig) M and G after recovery. RESULTS: From 1 January to 16 April 2020, 35 of 6001 PLWH experienced COVID-19, with a cumulative incidence of COVID-19 of 0.58% (95% confidence interval [CI], .42-.81%). Among the COVID-19 cases, 15 (42.86) had severe illness, with 2 deaths. The incidence, case-severity, and case-fatality rates of COVID-19 in PLWH were comparable to those in the entire population in Wuhan. There were 197 PLWH who had discontinued combination antiretroviral therapy (cART), 4 of whom experienced COVID-19. Risk factors for COVID-19 were age ≥50 years old and cART discontinuation. The median duration of SARS-CoV-2 viral shedding among confirmed COVID-19 cases in PLWH was 30 days (interquartile range, 20-46). Cases with high HIV viral loads (≥20 copies/mL) had lower IgM and IgG levels than those with low HIV viral loads (<20 copies/ml; median signal value divided by the cutoff value [S/CO] for IgM, 0.03 vs 0.11, respectively [P < .001]; median S/CO for IgG, 10.16 vs 17.04, respectively [P = .069]). CONCLUSIONS: Efforts are needed to maintain the persistent supply of antiretroviral treatment to elderly PLWH aged 50 years or above during the COVID-19 epidemic. The coinfection of HIV and SARS-CoV-2 might change the progression and prognosis of COVID-19 patients in PLWH.

Seroprevalence and humoral immune durability of anti-SARS-CoV-2 antibodies in Wuhan, China: a longitudinal, population-level, cross-sectional study.

BACKGROUND: Wuhan was the epicentre of the COVID-19 outbreak in China. We aimed to determine the seroprevalence and kinetics of anti-SARS-CoV-2 antibodies at population level in Wuhan to inform the development of vaccination strategies. METHODS: In this longitudinal cross-sectional study, we used a multistage, population-stratified, cluster random sampling method to systematically select 100 communities from the 13 districts of Wuhan. Households were systematically selected from each community and all family members were invited to community health-care centres to participate. Eligible individuals were those who had lived in Wuhan for at least 14 days since Dec 1, 2019. All eligible participants who consented to participate completed a standardised electronic questionnaire of demographic and clinical questions and self-reported any symptoms associated with COVID-19 or previous diagnosis of COVID-19. A venous blood sample was taken for immunological testing on April 14-15, 2020. Blood samples were tested for the presence of pan-immunoglobulins, IgM, IgA, and IgG antibodies against SARS-CoV-2 nucleocapsid protein and neutralising antibodies were assessed. We did two successive follow-ups between June 11 and June 13, and between Oct 9 and Dec 5, 2020, at which blood samples were taken. FINDINGS: Of 4600 households randomly selected, 3599 families (78·2%) with 9702 individuals attended the baseline visit. 9542 individuals from 3556 families had sufficient samples for analyses. 532 (5·6%) of 9542 participants were positive for pan-immunoglobulins against SARS-CoV-2, with a baseline adjusted seroprevalence of 6·92% (95% CI 6·41-7·43) in the population. 437 (82·1%) of 532 participants who were positive for pan-immunoglobulins were asymptomatic. 69 (13·0%) of 532 individuals were positive for IgM antibodies, 84 (15·8%) were positive for IgA antibodies, 532 (100%) were positive for IgG antibodies, and 212 (39·8%) were positive for neutralising antibodies at baseline. The proportion of individuals who were positive for pan-immunoglobulins who had neutralising antibodies in April remained stable for the two follow-up visits (162 [44·6%] of 363 in June, 2020, and 187 [41·2%] of 454 in October-December, 2020). On the basis of data from 335 individuals who attended all three follow-up visits and who were positive for pan-immunoglobulins, neutralising antibody levels did not significantly decrease over the study period (median 1/5·6 [IQR 1/2·0 to 1/14·0] at baseline vs 1/5·6 [1/4·0 to 1/11·2] at first follow-up [p=1·0] and 1/6·3 [1/2·0 to 1/12·6] at second follow-up [p=0·29]). However, neutralising antibody titres were lower in asymptomatic individuals than in confirmed cases and symptomatic individuals. Although titres of IgG decreased over time, the proportion of individuals who had IgG antibodies did not decrease substantially (from 30 [100%] of 30 at baseline to 26 [89·7%] of 29 at second follow-up among confirmed cases, 65 [100%] of 65 at baseline to 58 [92·1%] of 63 at second follow-up among symptomatic individuals, and 437 [100%] of 437 at baseline to 329 [90·9%] of 362 at second follow-up among asymptomatic individuals). INTERPRETATION: 6·92% of a cross-sectional sample of the population of Wuhan developed antibodies against SARS-CoV-2, with 39·8% of this population seroconverting to have neutralising antibodies. Our durability data on humoral responses indicate that mass vaccination is needed to effect herd protection to prevent the resurgence of the epidemic. FUNDING: Chinese Academy of Medical Sciences & Peking Union Medical College, National Natural Science Foundation, and Chinese Ministry of Science and Technology. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

A novel One-pot rapid diagnostic technology for COVID-19.

Novel coronavirus disease (COVID-19) caused by SARS-CoV-2 is an ongoing global pandemic associated with high rates of morbidity and mortality. RT-qPCR has become the diagnostic standard for the testing of SARS-CoV-2 in most countries. COVID-19 diagnosis generally relies upon RT-qPCR-mediated identification of SARS-CoV-2 viral RNA, which is costly, labor-extensive, and requires specialized training and equipment. Herein, we established a novel one-tube rapid diagnostic approach based upon formamide and colorimetric RT-LAMP (One-Pot RT-LAMP) that can be used to diagnose COVID-19 without the extraction of specific viral RNA. The technique could visually detect SARS-CoV-2 within 45 min with a limit of detection of 5 copies per reaction in extracted RNA, and about 7.66 virus copies per µL in viral transport medium. The One-Pot RT-LAMP test showed a high specificity without cross-reactivity with 12 viruses including SARS-CoV, MERS-CoV, and human infectious influenza virus (H1N1/H3N2 of influenza A and B virus, ect. We validated this One-Pot RT-LAMP approach by its successful use for the analysis of 45 clinical nasopharyngeal swab samples, yielding results identical to those of traditional RT-qPCR analyses, while achieving good selectivity and sensitivity relative to a commercial RT-qPCR approach. As such, this One-Pot RT-LAMP technology may be a valid means of conducting high-sensitivity, low-cost and rapid SARS-CoV-2 identification without the extraction of viral RNA.

Brief Report: Virologic and Immunologic Outcomes for HIV Patients With Coronavirus Disease 2019.

BACKGROUND: To describe the virologic and immunologic outcomes among people living with HIV (PLHIV) coinfected with SARS-CoV-2. SETTING: Wuhan, China. METHODS: Thirty-five coinfected patients were identified by matching the reported cases in National Notifiable Infectious Disease Report system for COVID-19 and HIV in Wuhan by time of April 19, 2020. Questionnaire-based survey and follow-up with blood sample collection were used to obtain characteristics before COVID-19 and after recovery. Nonparametric Mann-Whitney U test, χ2, or Fisher exact test, Mcnemar test, and Wilcoxon test were conducted. RESULTS: Twenty of the 35 coinfected patients were identified as asymptomatic/mild/moderate COVID-19 (nonsevere group) and 15 were identified as severe/critical (severe group). The severe and nonsevere group had no differences in demographics, HIV baseline status, the intervals between last tests and follow-up tests for CD4+ cell count and HIV-1 viral load (all P > 0.05). Overall, there was a significantly increased number of coinfected patients with HIV-1 viral load ≥20 copies/mL after recovery (P = 0.008). The median viral load increased significantly after recovery in severe group (P = 0.034), whereas no significant change of HIV-1 viral load was observed in the nonsevere group. Limited change of CD4+ cell count was found (all P > 0.05). CONCLUSION: The coinfection of SARS-CoV-2 may put PLHIV at greater risk for HIV-1 viral rebound especially for severe/critical COVID-19, whereas it had limited impacts on CD4+ cell count. Whether continuous antiretroviral therapy against HIV infection would have significant impacts on CD4+ cell count among PLHIV coinfected with SARS-CoV-2 needs further research.

A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection.

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals.IMPORTANCE We developed a visual and rapid reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the S gene for SARS-CoV-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings.