A successful diagnostic case of Pneumocystis pneumonia by the loop-mediated isothermal amplification method in a patient with dermatomyositis

Pneumocystis pneumonia (PCP) can occur in patients with many causes of the immunocompromised state other than human immunodeficiency virus (HIV). It is quite difficult to diagnose PCP without HIV because there is no method for detecting Pneumocystis jirovecii. Thus, non-HIV PCP continues to have high mortality. Recently, loop-mediated isothermal amplification (LAMP) is becoming an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. We report a non-HIV PCP case successfully diagnosed by the LAMP method. It was previously reported that PCR in BALF specimens had been the most sensitive method in the diagnosis of PCP without HIV. The LAMP method would be more sensitive than conventional PCR and an effective tool in the early diagnosis of PCP.     

Detection of Mycoplasma pneumoniae by loop-mediated isothermal amplification (LAMP) assay and serology in pediatric community-acquired pneumonia

Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for treatment with effective antimicrobial agents without delay; however, this capacity has not yet been established in clinical practice. Recently, a novel nucleic acid amplification method termed loop-mediated isothermal amplification (LAMP) has been used to rapidly diagnose various infectious diseases. In this study, we prospectively evaluated the efficacy of the LAMP assay to rapidly diagnose M. pneumoniae pneumonia in clinical practice. Three hundred sixty-eight children (median age, 3.8 years; range, 0.1–14.3 years) admitted to our hospital between April 2009 and March 2010 for community-acquired pneumonia were enrolled in this study. We obtained throat swabs on admission to detect M. pneumoniae DNA and paired serum samples on admission and at discharge to assay M. pneumoniae antibody titers. M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or a fourfold or greater increase in antibody titer. Overall, 46 children (12.5% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia; of these, 27 (58.7%) were aged less than 6 years. Of the aforementioned 46 children, 38 (82.6%) and 37 (80.4%) were identified by LAMP and serology, respectively. When the results of serology were taken as the standard, the sensitivity and specificity and positive and negative predictive values of the LAMP assay were 78.4%, 97.3%, 76.3%, and 97.6%, respectively. We concluded the LAMP assay may be useful for rapid diagnosis of M. pneumoniae pneumonia.     

Early favipiravir treatment was associated with early defervescence in non-severe COVID-19 patients

IntroductionThe antiviral drug favipiravir has been shown to have in vitro antiviral activity against severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2). In this study, we investigated the clinical benefits and initiation of favipiravir treatment in patients with non-severe coronavirus-disease-2019 (COVID-19).MethodsThis study was a single-center retrospective cohort study. Receiver operating characteristic curves were drawn to calculate the area under the curve, and the optimal cut-off values for the time to initiate favipiravir treatment were calculated to predict defervescence within seven days. Univariate and multivariate Cox regression analyses were performed to identify potential influencing factors of defervescence. This was defined as a body temperature of less than 37 °C for at least 2 days.ResultsData from 41 patients were used for the efficacy assessment. The days from the onset of fever to defervescence showed a positive correlation with the duration from the onset of fever to initiation of favipiravir treatment (r = 0.548, P < 0.001). The optimal cut-off value was the administration of favipiravir on day 4. Patients were assigned to two groups based on the optimal cut-off value from onset to initiation of favipiravir treatment: early treatment group (within 4-days) and late treatment group (more than 4-days). In the multivariate analysis, when adjusted for age, sex, and days from onset to initiation of favipiravir treatment, the significant factors were male sex and days of initiation of the favipiravir treatment.ConclusionsWe recommend that if favipiravir is to be used for treatment, it should be initiated as early as possible.     

A patient affected by critical COVID-19 pneumonia,successfully treated with convalescent plasma

We present a critically ill patient affected by COVID-19, whose chest computed tomography (CT) scan featured lung consolidations and severe patchy ground-glass opacitie. On day 3 since hospital admission the patient was placed on convalescent plasma treatment. A combined treatment with supportive care, hemoperfusion and convalescent plasma successfully managed to save the patient’s life. Convalescent plasma probably contributed to heal this patient and should always be considered in the management of critically ill COVID-19 cases.     

A study of quality assessment in SARS-CoV-2 pathogen nucleic acid amplification tests performance; from the results of external quality assessment survey of clinical laboratories in the Tokyo Metropolitan Government external quality assessment program in 2020

IntroductionThe Tokyo Metropolitan Government (TMG) conducted an external quality assessment (EQA) survey of pathogen nucleic acid amplification tests (NAATs) as a TMG EQA program for SARS-CoV-2 for clinical laboratories in Tokyo.MethodsWe diluted and prepared a standard product manufactured by Company A to about 2,500 copies/mL to make a positive control and distribute it with a negative control. The participants reported the use of the NAATs methods for SARS-CoV-2, the name of the real-time RT-PCR kit, the name of the detection device, the target gene(s), nucleic acid extraction kit, Threshold Cycle value in the case of RT-PCR and the Threshold time value and Differential calculation value in the case of Loop-Mediated Isothermal Amplification (LAMP) method.ResultsAs a result, 17 laboratories using fully automated equipment and 34 laboratories using the RT-PCR method reported generally appropriate results in this EQA survey. On the other hand, among the laboratories that adopted the LAMP method, there were a plurality of laboratories that judged positive samples to be negative.ConclusionThe false negative result is considered to be due to the fact that the amount of virus genome contained in the quality control reagent used this time was below the detection limit of the LAMP method combined with the rapid extraction reagent for influenza virus. On the other hand, false positive results are considered to be due to the non-specific reaction of the NAATs. The EQA program must be continued for the proper implementation of the pathogen NAATs.     

Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases

Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. This technology has been developed into commercially available detection kits for a variety of pathogens including bacteria and viruses. The current focus on LAMP methodology is as a diagnostic system to be employed in resource-limited laboratories in developing countries, where many fatal tropical diseases are endemic. The combination of LAMP and novel microfluidic technologies such as Lab-on-a-chip may facilitate the realization of genetic point-of-care testing systems to be used by both developed and developing countries in the near future. This review will describe the historical, current, and future developments of such technologies.     

Elevated blood favipiravir levels are inversely associated with ferritin levels and induce the elevation of uric acid levels in COVID-19 treatment: A retrospective single-center study

IntroductionMeasurement of blood Favipiravir (FPV) levels and accumulation of data in COVID-19 patients are critical for assessing FPV efficacy and safety. We performed a retrospective study based on measurements of blood levels of FPV and related factors in COVID-19 patients admitted to our hospital. Furthermore, we also investigated the association between blood FPV levels and uric acid level alterations before and after FPV administration.MethodsWe enrolled 27 COVID-19 patients who had received FPV treatment at Hokushin General Hospital from April 1 to December 31, 2020. Age, gender, COVID-19 severity, presence of comorbidities, and laboratory data for each subject were investigated to identify factors that correlate with blood FPV levels. Uric acid levels were measured before and after FPV administration and a difference between the levels (i.e., a change of uric acid level) was evaluated.ResultsWhen a significant univariate variable was input by the stepwise method and a combination of variables that maintained statistical superiority was searched, serum ferritin was the only factor that independently affected blood FPV level. Furthermore, in the high-FPV group (20 μg/mL or more), a significant increase in uric acid levels was observed after FPV administration. The increment value was significantly larger than that in the low-FPV group (less than 20 μg/mL).ConclusionsFerritin level was an important independent factor inversely affecting blood FPV level. Furthermore, a high blood FPV level induced the elevation of uric acid levels in COVID-19 treatment.     

Changes in diagnostic usefulness of the JRS scoring system in COVID-19 pneumonia by SARS-CoV-2 vaccination

IntroductionThe Japanese Respiratory Society (JRS) scoring system is a useful tool for the rapid presumptive diagnosis of atypical pneumonia in non-elderly (aged <60 years) patients. As SARS-CoV-2 vaccination progresses, COVID-19 in elderly people has markedly reduced. We investigated changes in diagnostic usefulness of the JRS scoring system in COVID-19 pneumonia between the Delta variant group (vaccination period) and non-Delta variant group (before the vaccination period).MethodsThis study was conducted at five institutions and assessed a total of 1121 patients with COVID-19 pneumonia (298 had the Delta variant). During the vaccination period, the Delta variant has spread and replaced the Alfa variant. We evaluated the vaccination period as the Delta variant group.ResultsAmong the six parameters of the JRS scoring system, matching rates of two parameters were higher in the Delta variant group than the non-Delta variant group (pre-vaccination period): age <60 years (77.5% vs 42.2%, P < 0.0001) and no or minor comorbid illness (69.1% vs 57.8%, p = 0.0007). The sensitivity of the diagnosis of atypical pneumonia in patients with COVID-19 pneumonia was significantly higher in the Delta variant group compared with the non-Delta variant group (80.2% vs 58.3%, p < 0.0001). When the diagnostic sensitivity was analyzed for different ages, the diagnostic sensitivities for the Delta variant and non-Delta variant groups were 92.6% and 95.5% for non-elderly patients and 39.1% and 32.5% for elderly patients, respectively.ConclusionsOur results demonstrated that the JRS scoring system is a useful tool for distinguishing between COVID-19 pneumonia and bacterial pneumonia in the COVID-19 vaccination period, but not before the vaccination period.     

A sporadic COVID-19 pneumonia treated with extracorporeal membrane oxygenation in Tokyo,Japan: A case report

IntroductionAn ongoing outbreak of a novel coronavirus disease (coronavirus disease 2019, COVID-19) has become a global threat. While clinical reports from China to date demonstrate that the majority of cases remain relatively mild and recover with supportive care, it is also crucial to be well prepared for severe cases warranting intensive care. Initiating appropriate infection control measures may not always be achievable in primary care or in acute-care settings.CaseA 45-year-old man was admitted to the intensive care unit due to severe pneumonia, later confirmed as COVID-19. His initial evaluation in the resuscitation room and treatments in the intensive care unit was performed under droplet and contact precaution with additional airborne protection using the N95 respirator mask. He was successfully treated in the intensive care unit with mechanical ventilation and extracorporeal membrane oxygenation for respiratory support; and antiretroviral treatment with lopinavir/ritonavir. His total intensive care unit stay was 15 days and was discharged on hospital day 24.ConclusionsStrict infection control precautions are not always an easy task, especially under urgent care in an intensive care unit. However, severe cases of COVID-19 pneumonia, or another novel infectious disease, could present at any moment and would be a continuing challenge to pursue appropriate measures. We need to be well prepared to secure healthcare workers from exposure to infectious diseases and nosocomial spread, as well as to provide necessary intensive care.     

A multi-stage SEIR(D) model of the COVID-19 epidemic in Korea

BackgroundThis paper uses a SEIR(D) model to analyse the time-varying transmission dynamics of the COVID-19 epidemic in Korea throughout its multiple stages of development. This multi-stage estimation of the model parameters offers a better model fit compared to the whole period analysis and shows how the COVID-19’s infection patterns change over time, primarily depending on the effectiveness of the public health authority’s non-pharmaceutical interventions (NPIs).MethodsThis paper uses the SEIR(D) compartment model to simulate and estimate the parameters for three distinctive stages of the COVID-19 epidemic in Korea, using a manually compiled COVID-19 epidemic dataset for the period between 18 February 2020 and 08 February 2021. The paper identifies three major stages of the COVID-19 epidemic, conducts multi-stage estimations of the SEIR(D) model parameters, and carefully infers context-dependent meaning of the estimation results to help better understand the unique patterns of the transmission of the novel coronavirus (SARS-CoV-2) in each stage.ResultsThe original SIR compartment model may produce a poor and even misleading estimation result if it is used to cover the entire period of the epidemic. However, if we use the model carefully in distinctive stages of the COVID-19 epidemic, we can find useful insights into the nature of the transmission of the novel coronavirus and the relative effectiveness of the government’s non-pharmaceutical interventions over time.

Key messages

• Identifies three distinctive waves of the COVID-19 epidemic in Korea.
• Conducts multi-stage estimations of the COVID-19 transmission dynamics using SEIR(D) epidemic models.
• The transmission dynamics of the COVID-19 vary over time, primarily depending on the relative effectiveness of the government’s non-pharmaceutical interventions (NPIs).
• The SEIR(D) epidemic model is useful and informative, but only when it is used carefully to account for the presence of multiple waves and context-dependent infection patterns in each wave.

     

SARS-CoV-2–specific antibody rearrangements in prepandemic immune repertoires of risk cohorts and patients with COVID-19

A considerable fraction of B cells recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with germline-encoded elements of their B cell receptor, resulting in the production of neutralizing and nonneutralizing antibodies. We found that antibody sequences from different discovery cohorts shared biochemical properties and could be retrieved across validation cohorts, confirming the stereotyped character of this naive response in coronavirus disease 2019 (COVID-19). While neutralizing antibody sequences were found independently of disease severity, in line with serological data, individual nonneutralizing antibody sequences were associated with fatal clinical courses, suggesting detrimental effects of these antibodies. We mined 200 immune repertoires from healthy individuals and 500 repertoires from patients with blood or solid cancers — all acquired prior to the pandemic — for SARS-CoV-2 antibody sequences. While the largely unmutated B cell rearrangements occurred in a substantial fraction of immune repertoires from young and healthy individuals, these sequences were less likely to be found in individuals over 60 years of age and in those with cancer. This reflects B cell repertoire restriction in aging and cancer, and may to a certain extent explain the different clinical courses of COVID-19 observed in these risk groups. Future studies will have to address if this stereotyped B cell response to SARS-CoV-2 emerging from unmutated antibody rearrangements will create long-lived memory.     

Coronavirus disease (COVID-19) associated delayed-onset fulminant myocarditis in patient with a history of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection

A healthy 35-year-old man was admitted to a rural hospital with coronavirus disease (COVID-19). During 14 days of hospitalization, he had no symptoms and was not given supplemental oxygen. About 3 weeks after discharge, he was re-admitted to the same hospital with new-onset continuous fever and general weakness. At the time of his second admission, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RT-PCR was performed on a retro-nasal swab and the result was negative. Four days after admission, the patient was transferred to our intensive care unit (ICU) following deterioration of his respiratory and haemodynamic conditions, where he received mechanical ventilation, intra-aortic balloon pumping, and veno-arterial extracorporeal membrane oxygenation. A nasopharyngeal swab was obtained again at ICU admission, but RT-PCR was negative for SARS-CoV-2. All antibody titres measured against other viruses were low. Blood cultures were negative, and no bacteria were observed in sputum samples. However, SARS-CoV-2 RNA was detected by RT-PCR from sections obtained by myocardial biopsy. The patient's final diagnosis was delayed-onset SARS-CoV-2-induced fulminant myocarditis (FM). We strongly suggested that one of the proposed mechanisms of COVID-19-related myocardial injury will be the direct invasion of SARS-CoV-2 into cardiomyocytes even if delayed-onset. And this is the first case of delayed-onset FM in which diagnosis of active myocarditis was proven by pathological examination following endomyocardial biopsy and SARS-CoV-2 was detected in the myocardium by RT-PCR.     

Specific detection of Angiostrongylus cantonensis in the snail Achatina fulica using a loop-mediated isothermal amplification (LAMP) assay

Angiostrongylus cantonensis, a rat lungworm, can cause eosinophilic meningitis and angiostrongyliasis in humans following ingestion of contaminated foods or intermediate/paratenic hosts with infective larvae. The snail Achatina fulica is one of the important intermediate hosts of A. cantonensis and is commonly eaten by humans in some countries. In the present study, we developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of A. cantonensis in Ac. fulica. Primers for LAMP were designed based on the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) of A. cantonensis. Specificity tests showed that only the products of A. cantonensis were detected when DNA samples of A. cantonensis and the heterologous control samples Anisakis simplex s.s, Trichuris trichiura, Toxocara canis, Trichinella spiralis and Ascaris lumbricoides were amplified by LAMP. Sensitivity evaluation indicated that the LAMP assay is 10 times more sensitive than the conventional polymerase chain reaction (PCR) assay. The established LAMP assay is rapid, inexpensive and easy to be performed. It can be used in clinical applications for rapid and sensitive detection of A. cantonensis in snails, which has implications for the effective control of angiostrongyliasis.     

Using high sensitivity cardiac troponin values in patients with SARS-CoV-2 infection (COVID-19): The Padova experience

BackgroundThe spectrum of Coronavirus Disease 2019 (COVID-19) is broad and thus early appropriate risk stratification can be helpful. Our objectives were to define the frequency of myocardial injury using high-sensitivity cardiac troponin I (hs-cTnI) and to understand how to use its prognostic abilities.MethodsRetrospective study of patients with COVID-19 presenting to an Emergency Department (ED) in Italy in 2020. Hs-cTnI was sampled based on clinical judgment. Myocardial injury was defined as values above the sex-specific 99th percentile upper reference limits (URLs). Most data is from the initial hospital value.Results426 unique patients were included. Hs-cTnI was measured in 313 (73.5%) patients; 85 (27.2%) had myocardial injury at baseline. Patients with myocardial injury had higher mortality during hospitalization (hazard ratio = 9 [95% confidence interval (CI) 4.55–17.79], p < 0.0001). Multivariable analysis including clinical and laboratory variables demonstrated an AUC of 0.942 with modest additional value of hs-cTnI. Myocardial injury was associated with mortality in patients with low APACHE II scores (<13) [OR (95% CI): 4.15 (1.40, 14.22), p = 0.014] but not in those with scores > 13 [OR (95% CI): 0.48 (0.08, 2.65), p = 0.40]. Initial hs-cTnI < 5 ng/L identified 33% of patients that were at low risk with 97.8% sensitivity (95% CI 88.7, 99.6) and 99.2% negative predictive value. Type 1 myocardial infarction (MI) and type 2 MI were infrequent.Conclusionshs-cTnI at baseline is a significant predictor of mortality in COVID-19 patients. A value < 5 ng/L identified patients at low risk.     

No SARS-CoV-2 RNA detected in the convalescent plasma of COVID-19 patients with different disease severity

IntroductionConvalescent plasma transfusion (CPT), a potential therapy for coronavirus disease 2019 (COVID-19), requires strict quality control of the donor blood. Whether to confirm the disappearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (RNAemia) in convalescent donor blood or not is unclear. Reports recommending the proof of viral disappearance from the blood are controversial. Foreseeing CPT in treating COVID-19 patients in Japan, we investigated RNAemia in 100 convalescent donors with mild, moderate, and severe COVID-19.MethodsBetween April 30 and July 30, 2020, we measured RNAemia in the plasma samples of donors with resolved COVID-19. Data on patients’ demographics, comorbidities, pneumonia, treatment, and real-time polymerase chain reaction results for SARS-CoV-2 were collected. Date of onset of initial symptoms or date of positive testing (for asymptomatic patients) were self-reported by the patients. Disease severity was defined as: no, mild, moderate oxygen demand, or severe (requiring mechanical ventilation).ResultsOf 100 donors (58 males [58.0%]; median age, 47 [range 22–69] years) screened as of July 30, 2020, 77 (77.0%); 19 (19.0%); and 4 (4.0%) had mild, moderate, and severe disease, respectively. Median time between onset and testing was 68.5 (range, 21–167) days. SARS-CoV-2 RNA was not detected in any of the plasma samples.ConclusionRNAemia was not found in recovered COVID-19 patients at least 21, 27, and 57 days after the onset of mild, moderate, and severe symptoms. Our study may contribute to determining a suitable time for collecting convalescent plasma from COVID-19 patients and to future CPT use.