Enterovirus D68 in Viet Nam (2009-2015)

Background: Since 1962, enterovirus D68 (EV-D68) has been implicated in multiple outbreaks and sporadic cases of respiratory infection worldwide, especially in the USA and Europe with an increasing frequency between 2010 and 2014. We describe the detection, associated clinical features and molecular characterization of EV-D68 in central and southern Viet Nam between 2009 and 2015. Methods: Enterovirus/rhinovirus PCR positive respiratory or CSF samples taken from children and adults with respiratory/central nervous system infections in Viet Nam were tested by an EV-D68 specific PCR. The included samples were derived from 3 different observational studies conducted at referral hospitals across central and southern Viet Nam 2009 2015. Whole-genome sequencing was carried out using a MiSeq based approach. Phylogenetic reconstruction and estimation of evolutionary rate and recombination were carried out in BEAST and Recombination Detection Program, respectively. Results: EV-D68 was detected in 21/625 (3.4%) enterovirus/rhinovirus PCR positive respiratory samples but in none of the 15 CSF. All the EV-D68 patients were young children (age range: 11.8 – 24.5 months) and had moderate respiratory infections. Phylogenetic analysis suggested that the Vietnamese sequences clustered with those from Asian countries, of which 9 fell in the B1 clade, and the remaining sequence was identified within the A2 clade. One intra sub-clade recombination event was detected, representing the second reported recombination within EV-D68. The evolutionary rate of EV-D68 was estimated to be 5.12E -3 substitutions/site/year. Phylogenetic analysis indicated that the virus was imported into Viet Nam in 2008. Conclusions: We have demonstrated for the first time EV-D68 has been circulating at low levels in Viet Nam since 2008, associated with moderate acute respiratory infection in children. EV-D68 in Viet Nam is most closely related to Asian viruses, and clusters separately from recent US and European viruses that were suggested to be associated with acute flaccid paralysis

Optic nerve sheath ultrasound for the detection and monitoring of raised intracranial pressure in tuberculous meningitis

Background: Neurological complications of tuberculous meningitis (TBM) often lead to raised intracranial pressure (ICP) resulting in high morbidity and mortality. Measurement of optic nerve sheath diameter (ONSD) by point-of-care ultrasound may aid in the identification and management of raised ICP in TBM. Methods: From June 2017 to December 2019, 107 Vietnamese adults with TBM, enrolled in the ACT HIV or LAST ACT trials (NCT03092817; NCT03100786), underwent ONSD ultrasound at one or more of days 0,3,7,14,21 +/-30 after enrolment. Demographic data, TBM severity grade, HIV co-infection status, and clinical endpoints by 3 months were recorded. ONSD values were correlated with disease severity, baseline brain magnetic resonance imaging or computed tomography imaging, cerebrospinal fluid parameters and clinical endpoints. Results: 267 ONSD ultrasound scans were performed in 107 participants over the first 30 days of treatment, with measurements from 0.38-0.74cm. Paired baseline ONSD and brain imaging were performed in 63 participants. Higher baseline ONSD was associated with more severe disease and abnormal brain imaging (abnormal imaging 0.55cm vs 0.50cm normal imaging, p=0.01). Baseline median ONSD was significantly higher in participants who died by 3 months (0.56cm [15/72]) vs. participants who survived by 3 months (0.52cm [57/72]), p=0.02. Median ONSD was higher at all follow up time points in participants who died by 3 months. Conclusions: Higher ONSD was associated with increased disease severity, brain imaging abnormalities, and increased death by 3 months. ONSD ultrasound has a potential role as a non-invasive and affordable bedside tool for predicting brain pathology and death in TBM.</p

Xpert MTB/RIF Ultra versus Xpert MTB/RIF for the diagnosis of tuberculous meningitis: a prospective, randomised, diagnostic accuracy study

Background: Xpert MTB/RIF Ultra (Xpert Ultra) might have higher sensitivity than its predecessor, Xpert MTB/RIF (Xpert), but its role in tuberculous meningitis diagnosis is uncertain. We aimed to compare Xpert Ultra with Xpert for the diagnosis of tuberculous meningitis in HIV-uninfected and HIV-infected adults. Methods: In this prospective, randomised, diagnostic accuracy study, adults (≥16 years) with suspected tuberculous meningitis from a single centre in Vietnam were randomly assigned to cerebrospinal fluid testing by either Xpert Ultra or Xpert at baseline and, if treated for tuberculous meningitis, after 3–4 weeks of treatment. Test performance (sensitivity, specificity, and positive and negative predictive values) was calculated for Xpert Ultra and Xpert and compared against clinical and mycobacterial culture reference standards. Analyses were done for all patients and by HIV status. Findings: Between Oct 16, 2017, and Feb 10, 2019, 205 patients were randomly assigned to Xpert Ultra (n=103) or Xpert (n=102). The sensitivities of Xpert Ultra and Xpert for tuberculous meningitis diagnosis against a reference standard of definite, probable, and possible tuberculous meningitis were 47·2% (95% CI 34·4–60·3; 25 of 53 patients) for Xpert Ultra and 39·6% (27·6–53·1; 21 of 53) for Xpert (p=0·56); specificities were 100·0% (95% CI 92·0–100·0; 44 of 44) and 100·0% (92·6–100·0; 48 of 48), respectively. In HIV-negative patients, the sensitivity of Xpert Ultra was 38·9% (24·8–55·1; 14 of 36) versus 22·9% (12·1–39·0; eight of 35) by Xpert (p=0·23). In HIV co-infected patients, the sensitivities were 64·3% (38·8–83·7; nine of 14) for Xpert Ultra and 76·9% (49·7–91·8; ten of 13) for Xpert (p=0·77). Negative predictive values were 61·1% (49·6–71·5) for Xpert Ultra and 60·0% (49·0–70·0) for Xpert. Against a reference standard of mycobacterial culture, sensitivities were 90·9% (72·2–97·5; 20 of 22 patients) for Xpert Ultra and 81·8% (61·5–92·7; 18 of 22) for Xpert (p=0·66); specificities were 93·9% (85·4–97·6; 62 of 66) and 96·9% (89·5–91·2; 63 of 65), respectively. Six (22%) of 27 patients had a positive test by Xpert Ultra after 4 weeks of treatment versus two (9%) of 22 patients by Xpert. Interpretation: Xpert Ultra was not statistically superior to Xpert for the diagnosis of tuberculous meningitis in HIV-uninfected and HIV-infected adults. A negative Xpert Ultra or Xpert test does not rule out tuberculous meningitis. New diagnostic strategies are urgently required.<br/

The influence of Strongyloides stercoralis co-infection on the presentation, pathogenesis and outcome of tuberculous meningitis

Background Helminth infections may modulate the inflammatory response to Mycobacterium tuberculosis and influence disease presentation and outcome. Strongyloides stercoralis is common amongst populations with high tuberculosis prevalence. Our aim was to determine if S. stercoralis co-infection influenced clinical presentation, cerebrospinal fluid (CSF) inflammation, and outcome from tuberculous meningitis (TBM). Methods From June 2017 to December 2019, 668 Vietnamese adults with TBM, enrolled in the ACT HIV or LAST ACT trials (NCT03092817; NCT03100786), underwent pre-treatment S. stercoralis testing by serology, stool microscopy, and/or stool PCR. Comparisons of pre-treatment TBM severity, CSF inflammation (including cytokines), and 3-month clinical endpoints were performed in active S. stercoralis infected and uninfected groups. Results Overall, 9.4% (63/668) participants tested positive for S. stercoralis. Active S. stercoralis infection was significantly associated with reduced pre-treatment CSF neutrophils (3 cells/mm 3[0-25] vs. 14 (cells/mm 3[1-83], p=0.04), and with reduced CSF IFN-ɣ, IL-2, and TNF-α concentrations (11.4 vs. 56.0pg/mL p=0.01; 33.1 vs. 54.5pg/mL p=0.03; 4.5 vs. 11.9pg/mL p=0.02, respectively), compared with uninfected participants. Neurological complications by 3 months were significantly reduced in active S. stercoralis infection vs. uninfected participants (3.8%[1/26] vs. 30.0%[33/110], respectively, p=0.01). Conclusions S. stercoralis co-infection may modulate the intracerebral inflammatory response to M. tuberculosis and improve TBM clinical outcomes

Central nervous system infection diagnosis by next-generation sequencing: a glimpse into the future?

Japanese encephalitis virus was detected by deep sequencing for the first time in urine of a 16-year-old boy with encephalitis. Seroconversion and polymerase chain reaction analysis confirmed the metagenomics finding. Urine is useful for diagnosis of flaviviral encephalitis, whereas deep sequencing can be a panpathogen assay for the diagnosis of life-threatening infectious diseases

Performance of metagenomic next-generation sequencing for the diagnosis of viral meningoencephalitis in a resource-limited setting

Background Meningoencephalitis is a devastating disease worldwide. Current diagnosis fails to establish the cause in ≥50% of patients. Metagenomic next-generation sequencing (mNGS) has emerged as pan-pathogen assays for infectious diseases diagnosis, but few studies have been conducted in resource-limited settings. Methods We assessed the performance of mNGS in the cerebrospinal fluid (CSF) of 66 consecutively treated adults with meningoencephalitis in a tertiary referral hospital for infectious diseases in Vietnam, a resource-limited setting. All mNGS results were confirmed by viral-specific polymerase chain reaction (PCR). As a complementary analysis, 6 viral PCR-positive samples were analyzed using MinION-based metagenomics. Results Routine diagnosis could identify a virus in 15 (22.7%) patients, including herpes simplex virus (HSV; n = 7) and varicella zoster virus (VZV; n = 1) by PCR, and mumps virus (n = 4), dengue virus (DENV; n = 2), and Japanese encephalitis virus (JEV; n = 1) by serological diagnosis. mNGS detected HSV, VZV, and mumps virus in 5/7, 1/1, and 1/4 of the CSF positive by routine assays, respectively, but it detected DENV and JEV in none of the positive CSF. Additionally, mNGS detected enteroviruses in 7 patients of unknown cause. Metagenomic MinION-Nanopore sequencing could detect a virus in 5/6 PCR-positive CSF samples, including HSV in 1 CSF sample that was negative by mNGS, suggesting that the sensitivity of MinION is comparable with that of mNGS/PCR. Conclusions In a single assay, metagenomics could accurately detect a wide spectrum of neurotropic viruses in the CSF of meningoencephalitis patients. Further studies are needed to determine the value that real-time sequencing may contribute to the diagnosis and management of meningoencephalitis patients, especially in resource-limited settings where pathogen-specific assays are limited in number

Central nervous system infection diagnosis by next-generation sequencing: a glimpse into the future?

Japanese encephalitis virus was detected by deep sequencing for the first time in urine of a 16-year-old boy with encephalitis. Seroconversion and polymerase chain reaction analysis confirmed the metagenomics finding. Urine is useful for diagnosis of flaviviral encephalitis, whereas deep sequencing can be a panpathogen assay for the diagnosis of life-threatening infectious diseases

Enterovirus serotypes in patients with central nervous system and respiratory infections in Viet Nam 1997-2010

Background: Enteroviruses are the most common causative agents of human illness. Enteroviruses have been associated with regional and global epidemics, recently, including with severe disease (Enterovirus A71 and D68), and are of interest as emerging viruses. Here, we typed Enterovirus A-D (EV) from central nervous system (CNS) and respiratory infections in Viet Nam. Methods: Data and specimens from prospective observational clinical studies conducted between 1997 and 2010 were used. Species and serotypes were determined using type-specific RT-PCR and viral protein 1 or 4 (VP1, VP4) sequencing. Results: Samples from patients with CNS infection (51 children – 10 CSF and 41 respiratory/rectal swabs) and 28 adults (28 CSF) and respiratory infection (124 children – 124 respiratory swabs) were analysed. Twenty-six different serotypes of the four Enterovirus species (A-D) were identified, including EV-A71 and EV-D68. Enterovirus B was associated with viral meningitis in children and adults. Hand, foot and mouth disease associated Enteroviruses A (EV-A71 and Coxsackievirus [CV] A10) were detected in children with encephalitis. Diverse serotypes of all four Enterovirus species were found in respiratory samples, including 2 polio-vaccine viruses, but also 8 CV-A24 and 8 EV-D68. With the exception of EV-D68, the relevance of these viruses in respiratory infection remains unknown. Conclusion: We describe the diverse spectrum of enteroviruses from patients with CNS and respiratory infections in Viet Nam between 1997 and 2010. These data confirm the global circulation of Enterovirus genera and their associations and are important for clinical diagnostics, patient management, and outbreak response.</p