Histological and Ultrastructural Changes in Lungs One Year Post Asymptomatic or Mild SARS-CoV-2 Infection: Evaluating the Potential for Active Virus Presence / Cambios Histológicos y Ultraestructurales en los Pulmones un Año Después de una Infección Asintomática o Leve por SARS-CoV-2: Evaluación del Potencial de Presencia Activa del Virus

SUMMARY: Prior research on post-COVID-19 or long COVID primarily focused on the presence of SARS-CoV-2 mostly in symptomatic patients. This study aimed to investigate the persistence of SARS-CoV-2 after 1 year of asymptomatic or mild COVID-19. SARS-CoV-2 infected and control K18-hACE2 transgenic mice (n=25) were studied. Moderate and severe symptomatic subjects were sacrificed after eight days, while mild or asymptomatic mice were kept in BSL-III for twelve months. Analyses included general condition, histochemistry, immunohistochemistry, transmission electron microscopy, and qRT-PCR. Lungs from the twelve-month group showed thickening of alveolar walls, with some lungs exhibiting the recruitment of inflammatory cells, the presence of SARS- CoV-2 mRNA, immunopositivity for the SARS-CoV-2 spike protein, and TEM showed viruses (60-125 nm) within vesicles, indicating continued replication. Certain lung samples showed persistent SARS-CoV-2 presence in Club cells, endothelial cells, and macrophages. The eight-day group exhibited viral interstitial pneumonitis, SARS-CoV-2 immunopositivity, and mRNA. The eight-day hearts displayed viral mRNA, while the twelve-month hearts tested negative. Some asymptomatic twelve-month subjects presented reduced surfactant, basal membrane thickening, fibrosis, and mild autonomic nerve degeneration. In this study conducted on mice, findings indicate the potential for chronic persistence of SARS-CoV-2 in the lungs one year post initial mild or asymptomatic infection, which could suggest the possibility of recurrent episodes in similar human conditions. The observed thickening of alveolar walls and potential fibrotic areas in these mice may imply an increased risk of post-COVID fibrosis in humans. Furthermore, the presence of SARS-CoV-2-positive inflammatory cells in some asymptomatic murine cases could herald a progression toward ongoing inflammation and chronic lung disease in humans. Therefore, the necessity for further studies in human subjects and vigilant monitoring of high-risk human populations is underscored.

Investigaciones anteriores sobre COVID-19 o COVID prolongado se centraron principalmente en la presencia de SARS-CoV-2 principalmente en pacientes sintomáticos. Este estudio tuvo como objetivo investigar la persistencia del SARS-CoV-2 después de 1 año de COVID-19 asintomático o leve. Se estudiaron ratones transgénicos K18-hACE2 infectados con SARS-CoV-2 y de control (n=25). Los animales con síntomas moderados y graves se sacrificaron después de ocho días, mientras que los ratones con síntomas leves o asintomáticos se mantuvieron en BSL-III durante doce meses. Los análisis incluyeron estado general, histoquímica, inmunohistoquímica, microscopía electrónica de transmisión y qRT- PCR. Los pulmones del grupo de doce meses mostraron engrosamiento de las paredes alveolares, y algunos pulmones exhibieron reclutamiento de células inflamatorias, presencia de ARNm del SARS-CoV-2, inmunopositividad para la proteína de la espícula del SARS-CoV-2 y TEM mostró virus (60 -125 nm) dentro de las vesículas, lo que indica una replicación continua. Ciertas muestras de pulmón mostraron una presencia persistente de SARS- CoV-2 en exocrinocitos bronquiolares, células endoteliales y macrófagos. El grupo de ocho días presentó neumonitis intersticial viral, inmunopositividad al SARS-CoV-2 y ARNm. Los corazones de ocho días mostraron ARNm viral, mientras que los corazones de doce meses dieron negativo. Algunos animales asintomáticos de doce meses presentaron disminución del surfactante, engrosamiento de la membrana basal, fibrosis y degeneración leve del nervio autónomo. En este estudio realizado en ratones, los hallazgos indican la posibilidad de persistencia crónica del SARS-CoV-2 en los pulmones un año después de la infección inicial leve o asintomática, lo que podría sugerir la posibilidad de episodios recurrentes en condiciones humanas similares. El engrosamiento observado de las paredes alveolares y las posibles áreas fibróticas en estos ratones puede implicar un mayor riesgo de fibrosis post-COVID en humanos. Además, la presencia de células inflamatorias positivas para SARS- CoV-2 en algunos casos murinos asintomáticos podría presagiar una progresión hacia una inflamación continua y una enfermedad pulmonar crónica en humanos. Por lo tanto, se subraya la necesidad de realizar más estudios en seres humanos y realizar un seguimiento atento de las poblaciones humanas de alto riesgo.

Exploration of an Efficient Simultaneous Molecular Detection Method of HIV, HCV, and Syphilis from a Single Dried Blood Spot / 生物医学与环境科学（英文）

Objective@#The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one dried blood spot (DBS) as an alternative sample to plasma.@*Method@#A total of 571 paired DBS/plasma samples were collected from men who have sex with men (MSM) and injection drug users (IDUs), and serological and molecular assays were performed. Using plasma results as the reference standard, the performance of DBS tests for HIV-1 RNA, HIV-1 DNA, and HCV RNA was evaluated. Pearson's correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma.@*Results@#Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA, five samples (5/32) were not detectable in DBS, while measurable HIV-1 RNA levels were present in plasma (1.44 to 3.99 log @*Conclusion@#The performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one DBS was acceptable. DBS, as an alternative sample to plasma, may be a viable option for the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access.

Pregnancy Outcomes in COVID-19: A Prospective Cohort Study in Singapore

INTRODUCTION@#Pregnant women are reported to be at increased risk of severe coronavirus disease 2019 (COVID-19) due to underlying immunosuppression during pregnancy. However, the clinical course of COVID-19 in pregnancy and risk of vertical and horizontal transmission remain relatively unknown. We aim to describe and evaluate outcomes in pregnant women with COVID-19 in Singapore.@*METHODS@#Prospective observational study of 16 pregnant patients admitted for COVID-19 to 4 tertiary hospitals in Singapore. Outcomes included severe disease, pregnancy loss, and vertical and horizontal transmission.@*RESULTS@#Of the 16 patients, 37.5%, 43.8% and 18.7% were infected in the first, second and third trimesters, respectively. Two gravidas aged ≥35 years (12.5%) developed severe pneumonia; one patient (body mass index 32.9kg/m2) required transfer to intensive care. The median duration of acute infection was 19 days; one patient remained reverse transcription polymerase chain reaction (RT-PCR) positive >11 weeks from diagnosis. There were no maternal mortalities. Five pregnancies produced term live-births while 2 spontaneous miscarriages occurred at 11 and 23 weeks. RT-PCR of breast milk and maternal and neonatal samples taken at birth were negative; placenta and cord histology showed non-specific inflammation; and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immunoglobulins were elevated in paired maternal and umbilical cord blood (n=5).@*CONCLUSION@#The majority of COVID-19 infected pregnant women had mild disease and only 2 women with risk factors (obesity, older age) had severe infection; this represents a slightly higher incidence than observed in age-matched non-pregnant women. Among the women who delivered, there was no definitive evidence of mother-to-child transmission via breast milk or placenta.

Presencia de trombosis y hallazgos anatomopatológicos post mortem en 109 pacientes con COVID-19 / Presence of thrombosis and post-mortem pathological findings in 109 patients with COVID-19

INTRODUCCIÓN: En 6 meses se notificaron más de 400 mil fallecidos por COVID-19. Han surgido múltiples investigaciones para comprender su etiopatogenia, siendo la autopsia médica uno de los mejores procedimientos para obtener información. Presentamos una revisión respecto a hallazgos post mortem publicados hasta mayo, 2020. RESULTADOS: Se recolectaron 12 estudios, de un total de 109 pacientes cuyo deceso fue por complicación respiratoria, predominó el sexo masculino, edad avanzada y con múltiples comorbilidades. El estudio PCR se realizó principalmente para diagnóstico. Se demostró ARN viral en riñón, hígado, corazón, cerebro y otros órganos. Los autores relataron presencia de micro y/o macro trombosis, en 50 de 109 casos, sobre todo a nivel pulmonar y renal, de tipo microscópica y relacionados a signos de shock. Desde la perspectiva anatomopatológica, se centra en alteraciones pulmonares y renales: daño alveolar difuso, injuria tubular aguda, microtrombos y otros signos de alteración microcirculatoria. Los estudios inmunohistoquímicos, de inmunofluoresencia y microscopía electrónica sugieren tropismo del virus por células epiteliales y estromales a nivel pulmonar y renal. En otros órganos se encuentran elementos morfológicos inespecíficos, atribuibles a patologías de base o shock. CONCLUSIÓN: El patrón histopatológico de daño alveolar difuso es frecuente, principalmente en fase exudativa o temprana. En el tejido renal destaca la injuria tubular aguda y daño microcirculatorio. El número y la descripción de muestras en otros órganos es reducida, siendo necesaria mayor casuística. La trombosis, es un trastorno prevalente en pulmones y riñones de pacientes con signos de shock. El tipo de trombo con más frecuencia descrito, es el microtrombo. Si bien se puede explicar como gatillante del fenómeno trombótico la interacción entre agente y huésped, otros factores deben ser estudiados para dilucidar la patogenia.

Identification of Zika virus in immature phases of Aedes aegypti and Aedes albopictus: a surveillance strategy for outbreak anticipation

A progressive increase in the circulation of arboviruses in tropical countries has been observed, accounting for 700,000 yearly deaths in the world. The main objective of this article was to identify the presence of Zika (ZIKV), dengue (DENV), and Chikungunya (CHIKV) viruses in immature stages of Aedes aegypti and Ae. albopictus. Household collections of immature phases of the vectors were carried out in the years 2015 and 2016. A total of 2902 dwellings were visited and the rate of infestation with larvae and pupae of Aedes mosquitoes was 283/1462 (19.4%) in March 2015 and 55/1440 (3.8%) in June 2015. In March 2015, 907 larvae/pupae were collected (583 or 64.3% of Ae. aegypti and 324 or 35.7% of Ae. albopictus) while in June 2015 there was a reduction in the number of immature forms found: 197 larvae/pupae (121 or 61.4% of Ae. aegypti and 76 or 38.6% of Ae. albopictus). This reduction was accompanied by a decrease in suspected human ZIKV cases from March to June 2015. The RT-qPCR performed in 18 pools identified that three (two of Ae. aegypti and one of Ae. albopictus) were positive for ZIKV, and none were positive for DENV or CHIKV. Our findings demonstrated that ZIKV was present in immature stages of insect vectors in the study region at least five months prior to the peak of ZIKV associated cases. Xenomonitoring of immature phases of the vectors may prove useful for predicting outbreaks.

Transcription and regulation of hepatitis B virus genes in host sperm cells / 亚洲男科学杂志(英文版)

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.

Detección del virus del dengue en larvas y pupas de Aedes aegypti recolectadas en áreas rurales del municipio de Anapoima, Cundinamarca, Colombia / Dengue virus detection in Aedes aegypti larvae and pupae collected in rural areas of Anapoima, Cundinamarca, Colombia

Resumen Introducción. La incidencia y la prevalencia del dengue en Cundinamarca son elevadas y, recientemente, se detectó Aedes aegypti en algunas áreas rurales del departamento. Objetivo. Evaluar la transmisión transovárica del virus del dengue en larvas y pupas recolectadas en áreas rurales del municipio de Anapoima. Materiales y métodos. Se recolectaron ejemplares vivos en 53 viviendas y se transportaron al laboratorio de Anapoima, donde se clasificaron, se agruparon y se congelaron. Llevadas a Bogotá, se las homogeneizó, se les extrajo el ARN con Trizol ® , se las sometió a una reacción en cadena de la polimerasa de transcripción inversa (Reverse Transcription Polymerase change reaction, RT-PCR) y a PCR convencional, y los productos amplificados se analizaron en geles de agarosa al 2 %. Resultados. En 54,7 % de las viviendas evaluadas se encontraron formas inmaduras del vector y el serotipo más frecuente fue el DENV-1. Sin embargo, en algunos pools se detectó la presencia simultánea de los serotipos DENV 1 y 2, DENV 1 y 3, y DENV 1 y 4, así como los serotipos DENV 1, 2 y 3. Conclusión. Los resultados confirmaron la transmisión vertical del virus de manera natural en el área rural del municipio, lo cual reafirma la capacidad vectorial de A. aegypti y explica, en parte, la persistencia del virus en la región y la posibilidad de que en la fase adulta el vector lo transmita sin haber consumido sangre infectada. Esta situación aumenta el riesgo de infección por el virus del dengue en Colombia y, por lo tanto, la necesidad de adelantar programas de prevención y control en todas las zonas con presencia del mosquito.

Abstract Introduction: There is a high incidence and prevalence of dengue in the department of Cundinamarca, and recently Aedes aegypti, the main vector of dengue virus (DENV), was detected in some of its rural areas. Objective: To evaluate viral transovarial transmission in larvae and pupae collected in rural areas of the municipality of Anapoima, Cundinamarca. Materials and methods: Live larvae and pupae were collected from 53 homes and later they were taken to the laboratory in Anapoima, where they were classified, pooled and frozen. In Bogotá, they were homogenized, RNA was extracted with Trizol™ , and RT-PCR and conventional PCR were performed. The amplified products were analyzed on 2% agarose gels. Results: In 54.7% of the houses we found A. aegypti in immature stages, and DENV-1 was the most frequent serotype. However, the simultaneous presence of DENV 1 and 2, DENV 1 and 3, DENV 1 and 4, and DENV 1, 2 and 3 serotypes was detected in some pools. Conclusion: The results confirmed the natural vertical transmission of the virus in the rural area under study. These findings confirmed the vector capacity of A. aegypti, and partly explains the persistence of the virus in the region and the possibility of transmission by the vector during adulthood without having ingested infected blood. This situation increases the risk of DENV infection in Colombia and the need for prevention and control programs in all areas where the mosquito is present.

Sensitivity and detection of chikungunya viral genetic material using several PCR-based approaches

Abstract INTRODUCTION: Chikungunya fever is a condition resulting from infection by chikungunya virus (CHIKV), an Aedes sp.-transmitted virus. This disease has been diagnosed in thousands of cases in the Americas, particularly in Brazil, in recent years, and there is an ongoing epidemic of chikungunya fever in Brazil that began in 2014. Clinical diagnosis is difficult; only a few cases have been confirmed by laboratory tests due to the low number of specific, efficient tests available for virus or antibody detection. Here, we aimed to evaluate different polymerase chain reaction (PCR) approaches for detection of CHIKV genetic material. METHODS: Specific primers and probes within the viral capsid gene region were designed for this work. To evaluate the analytic sensitivity of detection, human sera were spiked with serial dilutions of the viral stock. Several PCR protocols were performed to investigate the sensitivity of CHIKV RNA detection in serum dilutions ranging from 106 to 1 PFU equivalents. RESULTS: The technique showing the greatest sensitivity was a real-time PCR assay using specific probes that could detect the genetic material of the virus at all dilutions, followed by conventional PCR. Digital PCR showed low sensitivity and was much more expensive than other technologies. Digital PCR should be used for specific purposes other than clinical diagnosis. CONCLUSIONS: Although quantitative PCR using probes was more expensive than the use of intercalating dyes or conventional PCR, it had the highest sensitivity out of all tested PCR approaches.

High frequency of hepatitis E virus infection in swine from South Brazil and close similarity to human HEV isolates

Abstract Hepatitis E virus is responsible for acute and chronic liver infections worldwide. Swine hepatitis E virus has been isolated in Brazil, and a probable zoonotic transmission has been described, although data are still scarce. The aim of this study was to investigate the frequency of hepatitis E virus infection in pigs from a small-scale farm in the rural area of Paraná State, South Brazil. Fecal samples were collected from 170 pigs and screened for hepatitis E virus RNA using a duplex real-time RT-PCR targeting a highly conserved 70 nt long sequence within overlapping parts of ORF2 and ORF3 as well as a 113 nt sequence of ORF2. Positive samples with high viral loads were subjected to direct sequencing and phylogenetic analysis. hepatitis E virus RNA was detected in 34 (20.0%) of the 170 pigs following positive results in at least one set of screening real-time RT-PCR primers and probes. The swine hepatitis E virus strains clustered with the genotype hepatitis E virus-3b reference sequences in the phylogenetic analysis and showed close similarity to human hepatitis E virus isolates previously reported in Brazil.

Zika virus damages the human placental barrier and presents marked fetal neurotropism

An unusually high incidence of microcephaly in newborns has recently been observed in Brazil. There is a temporal association between the increase in cases of microcephaly and the Zika virus (ZIKV) epidemic. Viral RNA has been detected in amniotic fluid samples, placental tissues and newborn and fetal brain tissues. However, much remains to be determined concerning the association between ZIKV infection and fetal malformations. In this study, we provide evidence of the transplacental transmission of ZIKV through the detection of viral proteins and viral RNA in placental tissue samples from expectant mothers infected at different stages of gestation. We observed chronic placentitis (TORCH type) with viral protein detection by immunohistochemistry in Hofbauer cells and some histiocytes in the intervillous spaces. We also demonstrated the neurotropism of the virus via the detection of viral proteins in glial cells and in some endothelial cells and the observation of scattered foci of microcalcifications in the brain tissues. Lesions were mainly located in the white matter. ZIKV RNA was also detected in these tissues by real-time-polymerase chain reaction. We believe that these findings will contribute to the body of knowledge of the mechanisms of ZIKV transmission, interactions between the virus and host cells and viral tropism.

Survey of Clinical Laboratory Practices for 2015 Middle East Respiratory Syndrome Coronavirus Outbreak in the Republic of Korea

BACKGROUND: It is crucial to understand the current status of clinical laboratory practices for the largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infections in the Republic of Korea to be well prepared for future emerging infectious diseases. METHODS: We conducted a survey of 49 clinical laboratories in medical institutions and referral medical laboratories. A short questionnaire to survey clinical laboratory practices relating to MERS-CoV diagnostic testing was sent by email to the directors and clinical pathologists in charge of the clinical laboratories performing MERS-CoV testing. The survey focused on testing volume, reporting of results, resources, and laboratory safety. RESULTS: A total of 40 clinical laboratories responded to the survey. A total of 27,009 MERS-CoV real-time reverse transcription PCR (rRT-PCR) tests were performed. Most of the specimens were sputum (73.5%). The median turnaround time (TAT) was 5.29 hr (first and third quartile, 4.11 and 7.48 hr) in 26 medical institutions. The median TAT of more than a half of the laboratories (57.7%) was less than 6 hr. Many laboratories were able to perform tests throughout the whole week. Laboratory biosafety preparedness included class II biosafety cabinets (100%); separated pre-PCR, PCR, and post-PCR rooms (88.6%); negative pressure pretreatment rooms (48.6%); and negative pressure sputum collection rooms (20.0%). CONCLUSIONS: Clinical laboratories were able to quickly expand their diagnostic capacity in response to the 2015 MERS-CoV outbreak. Our results show that clinical laboratories play an important role in the maintenance and enhancement of laboratory response in preparation for future emerging infections.

Isolation of Middle East Respiratory Syndrome Coronavirus from a Patient of the 2015 Korean Outbreak

During the 2015 outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in Korea, 186 persons were infected, resulting in 38 fatalities. We isolated MERS-CoV from the oropharyngeal sample obtained from a patient of the outbreak. Cytopathic effects showing detachment and rounding of cells were observed in Vero cell cultures 3 days after inoculation of the sample. Spherical virus particles were observed by transmission electron microscopy. Full-length genome sequence of the virus isolate was obtained and phylogenetic analyses showed that it clustered with clade B of MERS-CoV.

Genotyping Influenza Virus by Next-Generation Deep Sequencing in Clinical Specimens

Rapid and accurate identification of an influenza outbreak is essential for patient care and treatment. We describe a next-generation sequencing (NGS)-based, unbiased deep sequencing method in clinical specimens to investigate an influenza outbreak. Nasopharyngeal swabs from patients were collected for molecular epidemiological analysis. Total RNA was sequenced by using the NGS technology as paired-end 250 bp reads. Total of 7 to 12 million reads were obtained. After mapping to the human reference genome, we analyzed the 3-4% of reads that originated from a non-human source. A BLAST search of the contigs reconstructed de novo revealed high sequence similarity with that of the pandemic H1N1 virus. In the phylogenetic analysis, the HA gene of our samples clustered closely with that of A/Senegal/VR785/2010(H1N1), A/Wisconsin/11/2013(H1N1), and A/Korea/01/2009(H1N1), and the NA gene of our samples clustered closely with A/Wisconsin/11/2013(H1N1). This study suggests that NGS-based unbiased sequencing can be effectively applied to investigate molecular characteristics of nosocomial influenza outbreak by using clinical specimens such as nasopharyngeal swabs.

External Quality Assessment of MERS-CoV Molecular Diagnostics During the 2015 Korean Outbreak

BACKGROUND: The largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infection outside Middle East Asia in 2015 has necessitated the rapid expansion of laboratories that conduct MERS-CoV molecular testing in Korea, together with external quality assessment (EQA) to evaluate the assays used. METHODS: The EQA program consisted of two phases; self-validation and blind assessment. For the first EQA phase, in vitro transcribed upstream region of the envelope gene (upE) and the open reading frame (ORF)1a RNAs were used at a concentration of 1,000 copies/microL. The test panel for the second EQA phase consisted of RNA extracts from three samples, which were obtained from two MERS-CoV positive patients and one MERS-CoV negative patient. RESULTS: The first EQA phase results for 46 participants showed a linear relationship between the threshold cycle (CT) values of RNA materials and the logarithmic concentrations for both upE and ORF1a gene targets (R2=0.73 and 0.75, respectively). The mean CT value for each concentration was different depending on which commercial kit was used for the assay. Among the three commonly used kits, PowerChek MERS Real-Time PCR kit (KogeneBiotech, Korea) showed the lowest CT values at all concentrations of upE and most concentrations of ORF1a. The second EQA phase results for 47 participants were 100% correct for all tested samples. CONCLUSIONS: This EQA survey demonstrates that the MERS-CoV molecular testing performed in Korea during the 2015 outbreak is of robust capability. However, careful establishment and validation of a cut-off value are recommended to ensure good analytical sensitivity.

Korean Society for Laboratory Medicine Practice Guidelines for the Molecular Diagnosis of Middle East Respiratory Syndrome During an Outbreak in Korea in 2015

For two months between May and July 2015, a nationwide outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) occurred in Korea. On June 3, 2015, the Korean Society for Laboratory Medicine (KSLM) launched a MERS-CoV Laboratory Response Task Force (LR-TF) to facilitate clinical laboratories to set up the diagnosis of MERS-CoV infection. Based on the WHO interim recommendations, the Centers for Disease Control and Prevention of United States guidelines for MERS-CoV laboratory testing, and other available resources, the KSLM MERS-CoV LR-TF provided the first version of the laboratory practice guidelines for the molecular diagnosis of MERS-CoV to the clinical laboratories on June 12, 2015. The guidelines described here are an updated version that includes case definition, indications for testing, specimen type and protocols for specimen collection, specimen packing and transport, specimen handling and nucleic acid extraction, molecular detection of MERS-CoV, interpretation of results and reporting, and laboratory safety. The KSLM guidelines mainly focus on the molecular diagnosis of MERS-CoV, reflecting the unique situation in Korea and the state of knowledge at the time of publication.

Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR

BACKGROUND: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. METHODS: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). RESULTS: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). CONCLUSIONS: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.

Analytical and Clinical Validation of Six Commercial Middle East Respiratory Syndrome Coronavirus RNA Detection Kits Based on Real-Time Reverse-Transcription PCR

BACKGROUND: During the 2015 outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV), six different commercial MERS-CoV RNA detection kits based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) were available in Korea. We performed analytical and clinical validations of these kits. METHODS: PowerChek (Kogene Biotech, Korea), DiaPlexQ (SolGent, Korea), Anyplex (Seegene, Korea), AccuPower (Bioneer, Korea), LightMix (Roche Molecular Diagnostics, Switzerland), and UltraFast kits (Nanobiosys, Korea) were evaluated. Limits of detection (LOD) with 95% probability values were estimated by testing 16 replicates of upstream of the envelope gene (upE) and open reading frame 1a (ORF1a) RNA transcripts. Specificity was estimated by using 28 nasopharyngeal swabs that were positive for other respiratory viruses. Clinical sensitivity was evaluated by using 18 lower respiratory specimens. The sensitivity test panel and the high inhibition panel were composed of nine specimens each, including eight and six specimens that were positive for MERS-CoV, respectively. RESULTS: The LODs for upE ranged from 21.88 to 263.03 copies/reaction, and those for ORF1a ranged from 6.92 to 128.82 copies/reaction. No cross-reactivity with other respiratory viruses was found. All six kits correctly identified 8 of 8 (100%) positive clinical specimens. Based on results from the high inhibition panel, PowerChek and AccuPower were the least sensitive to the presence of PCR inhibition. CONCLUSIONS: The overall sensitivity and specificity of all six assay systems were sufficient for diagnosing MERS-CoV infection. However, the analytical sensitivity and detection ability in specimens with PCR inhibition could be improved with the use of appropriate internal controls.

First Imported Case of Zika Virus Infection into Korea

Since Zika virus has been spreading rapidly in the Americas from 2015, the outbreak of Zika virus infection becomes a global health emergency because it can cause neurological complications and adverse fetal outcome including microcephaly. Here, we report clinical manifestations and virus isolation findings from a case of Zika virus infection imported from Brazil. The patient, 43-year-old Korean man, developed fever, myalgia, eyeball pain, and maculopapular rash, but not neurological manifestations. Zika virus was isolated from his semen, and reverse-transcriptase PCR was positive for the virus in the blood, urine, and saliva on the 7th day of the illness but was negative on the 21st day. He recovered spontaneously without any neurological complications. He is the first case of Zika virus infection in Korea imported from Brazil.