Diagnostic accuracy of direct reverse transcription-polymerase chain reaction using guanidine-based and guanidine-free inactivators for SARS-CoV-2 detection in saliva samples.

This study aimed to evaluate diagnostic accuracy of SARS-CoV-2 RNA detection in saliva samples treated with a guanidine-based or guanidine-free inactivator, using nasopharyngeal swab samples (NPS) as referents. Based on the NPS reverse transcription-polymerase chain reaction (RT-PCR) results, participants were classified as with or without COVID-19. Fifty sets of samples comprising NPS, self-collected raw saliva, and saliva with a guanidine-based, and guanidine-free inactivator were collected from each group. In patients with COVID-19, the sensitivity of direct RT-PCR using raw saliva and saliva treated with a guanidine-based and guanidine-free inactivator was 100.0%, 65.9%, and 82.9%, respectively, with corresponding concordance rates of 94.3% (κ=88.5), 82.8% (κ=64.8), and 92.0% (κ=83.7). Among patients with a PCR Ct value of <30 in the NPS sample, the positive predictive value for the three samples was 100.0%, 80.0%, and 96.0%, respectively. The sensitivity of SARS-CoV-2 RNA detection was lower in inactivated saliva than in raw saliva and lower in samples treated with a guanidine-based than with a guanidine-free inactivator. However, in individuals contributing to infection spread, inactivated saliva showed adequate accuracy regardless of the inactivator used. Inactivators can be added to saliva samples collected for RT-PCR to reduce viral transmission risk while maintaining adequate diagnostic accuracy.

Investigation of the use of PCR testing prior to ship boarding to prevent the spread of SARS-CoV-2 from urban areas to less-populated remote islands.

Preventing coronavirus disease (COVID-19) outbreaks and the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from urban areas to less-populated remote islands, many of which may have weak medical systems, is an important issue. Here, we evaluated the usefulness of pre-boarding, saliva-based polymerase chain reaction (PCR) screening tests to prevent the spread of SARS-CoV-2 from Tokyo to the remote island of Chichijima. The infection rate on the island during the study period from September 1, 2020 to March 21, 2021 was 0.015% (2/13,446). Of the 8,910 individuals tested before ship boarding, seven tested positive for COVID-19 (PCR tests of saliva samples). One was confirmed positive by subsequent confirmatory nasopharyngeal swab testing. Based on the testing results, positive cases were denied entry onto the ship to prevent the spread of COVID-19 from Tokyo to Chichijima. This study demonstrated that implementing pre-boarding PCR screening tests is a useful strategy that can be applied to other remote islands with vulnerable medical systems.

Discrimination of antibody to herpes B virus from antibody to herpes simplex virus types 1 and 2 in human and macaque sera.

The antigenic cross-reactive characteristics of herpes B virus and herpes simplex virus (HSV) type 1 (HSV-1) and HSV-2 are responsible for false-positive diagnoses by serological assays in humans and macaques. In the present study, we developed a fluorometric indirect enzyme-linked immunosorbent assay (ELISA) with recombinant herpes B virus glycoprotein D (gD) and HSV-1 and HSV-2 gG (gG-1 and gG-2, respectively) to discriminate between the three primate herpesvirus infections. The secreted form of gD, gDdTM, was used to detect antibody to herpes B virus gD. Sera positive for herpes B virus, HSV-1, and HSV-2 showed specific reactions to gD, gG-1, and gG-2, respectively. Sera collected from humans and rhesus macaques were investigated for the presence of antibodies to the recombinant proteins of the three herpesviruses. The results suggested that the approach is able to discriminate between herpes B virus and HSV infections. The ELISA was also found to be able to detect infections with multiple primate herpesviruses and may have the potential to identify a subsequent infection in individuals that have already been infected with another herpesvirus. In addition, we found evidence of a greater cross-reactivity of herpes B virus with HSV-1 than with HSV-2. It is suggested that the ELISA with the recombinant antigens is useful not only for the serodiagnosis of primate herpesvirus infections but also for elucidation of the seroprevalence of herpesviruses in humans and primates.

Epstein-Barr virus load in cerebrospinal fluid of patients with chronic active Epstein-Barr virus infection.

Chronic active Epstein-Barr virus (EBV) infection is a rare chronic mononucleosis syndrome involving clonally proliferating EBV-infected T-/NK-cells. EBV DNA was quantified in nonpleocytotic cerebrospinal fluid (CSF) of 9 patients. Three patients with neurologic and/or neuroimaging abnormalities showed high CSF copy numbers. In 1 patient, CSF copy number exceeded the peripheral blood value. CSF EBV-load may predict the central nervous system involvement of EBVT-/NK-cells.

Japanese external quality assessment program to standardize HIV-1 drug-resistance testing (JEQS2010 program) using in vitro transcribed RNA as reference material.

To design appropriate antiretroviral therapy regimens and avoid the emergence of human immunodeficiency virus (HIV)-1 variants with reduced susceptibility to antiretroviral drugs, genotypic drug-resistance testing (HIV genotyping) is strongly recommended. To monitor the quality of HIV genotyping in Japan, we performed an external quality assessment (EQA), named the Japanese external quality assessment program, to standardize HIV genotyping (JEQS). To accurately evaluate the quality of HIV genotyping, we employed as reference material (RM) a well-characterized sample, in vitro transcribed RNA (trRNA) that includes the HIV gag-pol sequence, and created a JEQS2010 panel consisting of three single variant and three mixed trRNA samples. All 11 participating laboratories showed high concordance rates (>96%) for the single variant samples. Eight laboratories also showed good rates of detecting minor variants, but three laboratories failed to detect the variants comprising one-half of the sample. These three laboratories used a common primer that had four internal mismatches to the minor trRNA clone. This program showed the usefulness of trRNA as RM, the high quality of HIV genotyping, and extensive interlaboratory variation in the ability to detect minor variants. These results suggest that improving the quality of HIV genotyping in Japan requires regularly implementing the EQA program and improving the HIV genotyping protocol in each laboratory.

Chronic graft-versus-host disease following varicella-zoster virus infection in allogeneic stem cell transplant recipients.

We describe 2 allogeneic stem cell transplantation patients who developed chronic graft-versus-host disease (GVHD) after dermatomal varicella-zoster virus (VZV) infection. Localized zoster did not respond to oral valaciclovir but did resolve with intravenous aciclovir. However, skin eruptions, eye/oral dryness, and liver dysfunction were observed at the healing stage of localized zoster, suggesting development of GVHD. Intensification of immunosuppressive therapy was required to control GVHD. Quantitative real-time PCR for VZV DNA was used to distinguish liver involvement by chronic GVHD from visceral dissemination of VZV in 1 patient. VZV infection may trigger chronic GVHD after allogeneic stem cell transplantation.

Multiplex PCR for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila in community-acquired pneumonia.

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila. Oligonucleotide primers for the amplification of the DNA of these three organisms were optimized for use in combination in the same reaction. PCR products were detected by the Micro-Chip Electrophoresis Analysis System. Clinical samples were obtained from 208 community-acquired pneumonia (CAP) patients who were participants in a multicenter CAP surveillance study performed at seven medical schools and their affiliate hospitals in Japan. No significant differences in the sensitivity of each primer set were observed when tested in both the multiplex and monoplex PCR assays. Our multiplex PCR was able to reliably detect 10 copies/100 microl of each of the three pathogen DNAs. Of the panel of 208 samples, 14 of 15 C. pneumoniae, 10 of 10 M. pneumoniae, eight of eight L. pneumophila and 165 of 176 negative samples were correctly identified. Eleven cases who were the multiplex PCR positive and conventional method negative were observed. The PCR findings were of possible significance in at least four of these patients. Our multiplex PCR assay could potentially be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical usefulness.

Herpesvirus infections of the central nervous system.

In recent years, advances in the diagnosis and treatment of herpes simplex encephalitis (HSE) have been achieved due to the prevalence of antiviral drugs and the introduction of the polymerase chain reaction (PCR) to test the cerebrospinal fluid. The several clinical forms of herpes simplex virus type 1 (HSV-1) infections of the central nervous system (CNS), including acute disseminated encephalomyelitis and brainstem encephalitis, have been clarified. However, fatal, prolonged, or relapsed cases are still observed, and early detection and appropriate treatment is necessary to lead to a good prognosis for these intractable HSE cases. In adult HSV-2 infections, meningitis and myelitis associated with genital herpes are common. In the past, HSV-2 myelitis has been reported as a form of fatal necrotizing myelopathy; however, using PCR and magnetic resonance imaging studies, mild surviving cases are increasingly likely to be identified. Meanwhile, various CNS syndromes resulting from the herpes group viruses, including varicella-zoster virus and Epstein-Barr virus have also been reported. These herpesviruses have several characteristics in common, e.g., they exist in the latent state and they occur in both mucocutaneous and CNS infections. Adult HSV-1 and -2 infections of the CNS are discussed together with other herpes group virus infections of the CNS.

Recurrent herpes simplex virus type 2 meningitis: a case report of Mollaret's meningitis.

It is well known that herpes simplex virus (HSV) type 2 produces acute meningitis, while HSV type 2 rarely causes recurrent meningitis (Mollaret's meningitis). We report the history of a 40-year-old patient with recurrent HSV type 2 meningitis (Mollaret's meningitis). The patient had seven episodes of meningeal symptoms within a 7-year period. In the seventh episode, HSV type 2 DNA was confirmed by nested polymerase chain reaction (PCR) with the cerebrospinal fluid (CSF). A real-time quantitative PCR study of the first CSF sample detected 2,000 copies of the HSV genome, which rapidly disappeared following treatment with acyclovir. The present case may be the first case of HSV type 2 Mollaret's meningitis to be documented in Japan. In our case, HSV serum antibody titers were at low levels during the whole course of the disease. The possible pathophysiology of this case is discussed.

Clinical usefulness of multiplex PCR lateral flow in MRSA detection: a novel, rapid genetic testing method.

Methicillin-resistant Staphylococcus aureus (MRSA) with exogenous cassette DNA containing the methicillin-resistant gene mecA (SCCmec) poses a problem as a drug-resistant bacterium responsible for hospital- and community-acquired infections. The frequency of MRSA detection has recently been increasing rapidly in Japan, and SCCmec has also been classified more diversely into types I-V. A rapid test is essential for early diagnosis and treatment of MRSA infections, but detection by conventional methods requires at least two days. The newly developed multiplex PCR lateral flow method allows specific amplification of femA to detect S. aureus, mecA to detect SCCmec, and kdpC to detect SCCmec type II; moreover, PCR products can be evaluated visually in about 3 h. In the present study, we developed a PCR lateral flow method for MRSA using this method and investigated its clinical usefulness in the detection of MRSA. The results showed a diagnostic concordance rate of 91.7% for MRSA and methicillin-susceptible S. aureus between bacteriological examination and PCR lateral flow, and a high level of specificity in PCR lateral flow. In addition, a higher detection rate for S. aureus using the same sample was observed for PCR lateral flow (70.2%) than for bacteriological tests (48.6%). The above results show that PCR lateral flow for MRSA detection has high sensitivity, specificity, and speed, and its clinical application as a method for early diagnosis of MRSA infections appears to be feasible.

Sequential adenovirus infection of type 14 hemorrhagic cystitis and type 35 generalized infection after cord blood transplantation.

We report a case of a 29-year-old male patient with a generalized adenovirus (AdV) infection after cord blood transplantation (CBT) for acute myelocytic leukemia with maturation at 2nd complete remission. Before engraftment, hemorrhagic cystitis was caused by AdV, which resulted in hydronephrosis, renal failure, and adenoviremia on day 34. Forced diuresis, hemodialysis, withdrawal of cyclosporin A, and administration of gamma-globulin or vidarabine were not effective and the patient died of pulmonary alveolar hemorrhage on day 67. At autopsy, old inflammatory change only was observed in the bladder section. In the lungs and kidneys, granular deposits in the nucleus and a high copy number of AdV-DNA were observed. Molecular diagnosis using PCR-restriction fragment length polymorphism analysis demonstrated that AdV with the serotype 14 caused the cystitis. However, retrospective genome typing using PCR sequencing revealed the infection of AdV serotype 35 in the kidneys, lungs, and serum. The present case suggested that Adv infection could not be always caused by a single AdV serotype, and suggested that multiple serotype infection was very difficult to treat. It is desired that a consensus regarding the treatment of AdV infections is established.

Evaluation of a rapid immunochromatographic ODK0501 assay for detecting Streptococcus pneumoniae antigen in sputum samples from patients with lower respiratory tract infection.

A novel, rapid, and noninvasive test (ODK0501) to detect Streptococcus pneumoniae antigen was evaluated in a Japanese multicenter study. ODK0501 uses polyclonal antibodies to detect C polysaccharide of S. pneumoniae from sputum samples by an immunochromatographic assay. The utility of ODK0501 was evaluated for 161 adult patients with lower respiratory tract infection between March 2006 and March 2007. Bacterial culture and identification, real-time PCR, and ODK0501 assays were performed on sputum samples, and the Binax Now Streptococcus pneumoniae antigen test was performed using urine samples obtained from the same patients. The performances of all tests were compared based on the results of bacterial culture and identification. The sensitivity and specificity of ODK0501 were 89.1% (49/55 samples) and 95.3% (101/106 samples), respectively. We then compared the Binax Now Streptococcus pneumoniae antigen test with ODK0501 using samples from 142 patients. The sensitivities of ODK0501 and the Binax Now S. pneumoniae antigen test were 90.0% (45/50 samples) and 62.0% (31/50 samples), respectively (P = 0.002). The relative quantity of S. pneumoniae in expectorated sputum was calculated using real-time PCR and indicated that the possibility of false-positive results for ODK0501 due to indigenous S. pneumoniae was low. The positive and negative concordance rates of ODK0501 and Binax Now were 96.8% (30/31 samples) and 21.1% (4/19 samples), respectively. Binax Now was less capable of detecting S. pneumoniae antigen among patients with underlying chronic obstructive pulmonary disease. In conclusion, ODK0501 is noninvasive, rapid, and an accurate tool for diagnosing respiratory infection caused by S. pneumoniae.

Prevalence of Epstein-Barr virus in Japan: trends and future prediction.

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and some malignancies including EBV-associated-lymphomas. A large portion of adults all over the world are infected with EBV. In children, however, there are geographic variations. Most of the children in Asia and in other developing countries are infected in their early life, before 1 year of age (>90% of 5-9-year-old children are infected), while the age of primary infection is delayed in Western countries (approx. 50% of 5-9-year-old children are infected). The purpose of the present paper was to investigate the recent time trend of the EBV seropositivity among 5-7-year-old children living in Tokyo and its neighboring prefectures. Indirect immunofluorescein study for IgG antibody to viral capsid antigen was performed on 442 archival sera. Before the early 1990s, >80% of 5-7-year-old children were found to be seropositive, while the positivity rate decreased to 59% (P < 0.001) for the years 1995-1999. These results also showed that the delay in the age of primary infection is continuing and that the rate is estimated to be <50% in 2006. This result suggests that the delay will affect the incidence of EBV-associated disorders in Japan.

Immunoglobulin G antibody avidity assay for serodiagnosis of hepatitis C virus infection.

It has been reported that the avidity of specific IgG antibody is lower in primary viral infection than in chronic viral infection. However, few studies have been reported on the IgG avidity in hepatitis C virus (HCV) infection. In the present study, 36 patients with antibody to HCV (anti-HCV) were examined for IgG avidity by an enzyme immunoassay with or without urea elution. The avidity index was significantly low in patients with primary HCV infection (7.7 +/- 6.8%, mean +/- SD), compared with patients with chronic HCV infection (77.0 +/- 21.8%) and individuals with past HCV infection (44.5 +/- 12.6%). Temporal changes of IgG avidity were examined in six patients with primary HCV infection. The avidity index was low in the acute phase of the infection and then increased with time. These results suggest that the avidity assay for IgG anti-HCV is a useful method for distinguishing primary HCV infection from chronic or past HCV infection.

Dynamics of Epstein-Barr virus load in pyothorax-associated lymphoma.

We treated a 73-year-old man who developed a pyothorax-associated pleural lymphoma after a 52-year history of tuberculous pleuritis. The lymphoma was classified histologically as a diffuse large, B-cell type. Epstein-Barr virus (EBV) was identified in the tumor by immunocytochemical and molecular methods. Chemotherapy both without and with the addition of rituximab was only minimally effective, but adding radiotherapy to chemotherapy reduced the tumor size, resulting in a partial remission. The EBV load measured by a quantitative real-time polymerase chain reaction correlated well with the tumor size and the serum lactate dehydrogenase concentration. Monitoring the EBV load may be useful in the management of pyothorax-associated lymphoma.