High Prevalence of Coinfecting Enteropathogens in Suspected Rotavirus Vaccine Breakthrough Cases.

Despite the global use of rotavirus vaccines, vaccine breakthrough cases remain a pediatric health problem. In this study, we investigated suspected rotavirus vaccine breakthrough cases using next-generation sequencing (NGS)-based viral metagenomics (n = 102) and a panel of semiquantitative reverse transcription-PCR (RT-qPCR) (n = 92) targeting known enteric pathogens. Overall, we identified coinfections in 80% of the cases. Enteropathogens such as adenovirus (32%), enterovirus (15%), diarrheagenic Escherichia coli (1 to 14%), astrovirus (10%), Blastocystis spp. (10%), parechovirus (9%), norovirus (9%), Clostridioides (formerly Clostridium) difficile (9%), Dientamoeba fragilis (9%), sapovirus (8%), Campylobacter jejuni (4%), and Giardia lamblia (4%) were detected. Except for a few reassortant rotavirus strains, unusual genotypes or genotype combinations were not present. However, in addition to well-known enteric viruses, divergent variants of enteroviruses and nonclassic astroviruses were identified using NGS. We estimated that in 31.5% of the patients, rotavirus was likely not the cause of gastroenteritis, and in 14.1% of the patients, it contributed together with another pathogen(s) to disease. The remaining 54.4% of the patients likely had a true vaccine breakthrough infection. The high prevalence of alternative enteropathogens in the suspected rotavirus vaccine breakthrough cases suggests that gastroenteritis is often the result of a coinfection and that rotavirus vaccine effectiveness might be underestimated in clinical and epidemiological studies.

Comparison of four commercial SARS-CoV-2 IgG immuno-assays in RT-PCR negative patients with suspect CT findings.

A subset of patients with Covid-19 presents with negative RT-PCR screening but suspect CT findings. Using four commercially available anti-SARS-CoV-2 IgG immuno-assays, we found this subset constituted 9.2% of all consecutively admitted outpatients with Covid-19 in our hospital. Clinical specificity for Covid-19 of some N protein-based immuno-assays was suboptimal, as positive results were observed in control patients with recent common human coronavirus, influenza B and adenovirus infections.

Mycoplasma genitalium acquisition and macrolide resistance after initiation of HIV pre-exposure prophylaxis in men who have sex with men.

OBJECTIVES: Recent evidence shows that patients using HIV pre-exposure prophylaxis (PrEP) have an increased rate of bacterial STIs, including syphilis, chlamydia and gonorrhoea. Our study aimed to describe the acquisition and the susceptibility for macrolides of Mycoplasma genitalium in men who have sex with men (MSM) on PrEP. METHODS: We studied all MSM who started PrEP in the AZ Sint-Jan Hospital Bruges from 1 June 2017 to 31 March 2019 with at least one follow-up visit. Patients were screened for M. genitalium and other STIs with pooled rectal swabs, pharyngeal swabs and first-voided urine, and blood samples at baseline and quarterly intervals after initiating PrEP. TaqMan Array Card technology was used to detect M. genitalium and determine macrolide-resistance mediating mutations in region V of the 23S rRNA gene (A2058G, A2059G, A2058C and others). Patients with an STI were treated based on a national guideline. RESULTS: 131 MSM (median age 40 years, range 20-79) were included in the study. The median follow-up time was 12 months (IQR 6.1-17). Baseline prevalence of M. genitalium was 6.9% and incidence rate after PrEP initiation was 28.8 per 100 person-years (95% CI 21.7 to 37.2), without significant differences in proportions between the first four quarterly intervals. All but one acquisitions were asymptomatic. Younger age and positivity for M. genitalium at baseline were significantly associated with incident M. genitalium acquisition. The observed proportion of macrolide resistance increased not significantly from 44% at baseline to 57%-86% after PrEP initiation. None of the 27 macrolide-resistant M. genitalium acquisitions could be linked to azithromycin exposure in the three preceding months. CONCLUSIONS: After initiation of PrEP, we found a stable incidence of almost exclusively asymptomatic M. genitalium. However, a non-significant trend of an increased percentage of macrolide-resistant strains was observed.

What is the risk of missing legionellosis relying on urinary antigen testing solely? A retrospective Belgian multicenter study.

Currently, diagnosis of legionellosis relies mainly on urinary antigen testing (UAT) for Legionella pneumophila serogroup 1 (Lp1). However, this test has several limitations, particularly missing non-Lp1 infections. The purpose of this large multicenter study was to investigate the risk of missing legionellosis relying on UAT solely. Molecular results of Legionella detection as part of a first-line (syndromic) testing algorithm for severe respiratory tract infections were investigated retrospectively and compared with UAT results in 14 Belgian laboratories. Overall, 44.4% (20/45) UAT results appeared false negative and were reclassified as legionellosis based on PCR findings [Legionnaires' disease, 37.5% (15/40); Pontiac fever, 100% (5/5)]. A total of 39.4% (26/66) diagnosis probably would have been missed or delayed without a syndromic approach, as UAT or specific molecular testing for Legionella was not requested by the clinician. Furthermore, we confirmed the higher sensitivity of molecular Legionella detection in lower respiratory tract compared with upper respiratory tract specimens (p = 0.010).

Epidemiology and clinical impact of viral, atypical, and fungal respiratory pathogens in symptomatic immunocompromised patients: a two-center study using a multi-parameter customized respiratory Taqman® array card.

The prevalence of respiratory viruses in immunocompromised adult patients and the association with clinical outcomes is still underexplored. Our goal was to assess the epidemiology and the potential clinical impact of respiratory viral infections in a high-risk patient population. Two large hospitals performed a respiratory Taqman array card (TAC), targeting 24 viruses, 8 bacteria, and 2 fungi simultaneously, on 435 samples from 397 symptomatic immunocompromised patients. Clinical details were collected retrospectively using a structured case report form. An overall positivity rate of 68% was found (51% mono- and 17% co-infections). Pathogen distribution was as follows: influenza A (20.7%), rhinoviruses (15.2%), coronaviruses (7.8%), Pneumocystis jirovecii (7.4%), RSV (7.1%), and CMV (6.0%) were the most frequently encountered, followed by HSV (5.5%), hMPV (4.4%), parainfluenza viruses (3.9%), influenza B (3.7%), and Aspergillus species (3.7%). Other pathogens were not detected or detected only in ≤ 1% of samples. Hospital and ICU admission rates were 84% and 11%, respectively. The presence of a pathogen was strongly associated with higher need for supplemental oxygen (p = 0.001), but it had no impact on ICU admission, mechanical ventilation requirement, antibacterial therapy, or mortality. In conclusion, our study described the epidemiology of respiratory pathogens in a large group of symptomatic immunocompromised patients and provides evidence of a relationship between pathogen detection and the need for supplemental oxygen. This association was still found after the exclusion of the results positive for influenza viruses, suggesting that non-influenza viruses contribute to severe respiratory illness in patients with compromised immunity.

Urogenital pathogens, associated with Trichomonas vaginalis, among pregnant women in Kilifi, Kenya: a nested case-control study.

BACKGROUND: Screening of curable sexually transmitted infections is frequently oriented towards the diagnosis of chlamydia, gonorrhea, syphilis and trichomoniasis, whereas other pathogens, sometimes associated with similar urogenital syndromes, remain undiagnosed and/or untreated. Some of these pathogens are associated with adverse pregnancy outcomes. METHODS: In a nested case-control study, vaginal swabs from 79 pregnant women, i.e., 28 T. vaginalis-positive (cases) and 51 T. vaginalis-negative (controls), were screened by quantitative PCR for Adenovirus 1 and 2, Cytomegalovirus, Herpes Simplex Virus 1 and 2, Chlamydia trachomatis, Escherichia coli, Haemophilus ducreyi, Mycoplasma genitalium, M. hominis, candidatus M. girerdii, Neisseria gonorrhoeae, Streptococcus agalactiae, Treponema pallidum, Ureaplasma parvum, U. urealyticum, and Candida albicans. Additionally, we determined whether women with pathogens highly associated with T. vaginalis had distinct clinical signs and symptoms compared to women with T. vaginalis mono-infection. RESULTS: M. hominis was independently associated with T. vaginalis (adjusted odds ratio = 6.8, 95% CI: 2.3-19.8). Moreover, M. genitalium and Ca M. girerdii were exclusively detected in women with T. vaginalis (P = 0.002 and P = 0.001), respectively. Four of the six women co-infected with T. vaginalis and Ca M. girerdii complained of vaginal itching, compared to only 4 out of the 22 women infected with T. vaginalis without Ca M. girerdii (P = 0.020). CONCLUSION: We confirm M. hominis as a correlate of T. vaginalis in our population, and the exclusive association of both M. genitalium and Ca. M. girerdii with T. vaginalis. Screening and treatment of these pathogens should be considered.

Heads or Tails: Genotyping of Hepatitis C Virus Concerning the 2k/1b Circulating Recombinant Form.

As different hepatitis C virus (HCV) genotypes respond differently to initiated therapy, correct HCV genotyping is essential. A potential risk for misclassification of the intergenotypic HCV circulating recombinant form (CRF) 2k/1b strains exists, depending on the genotyping method used. The aim was to investigate the differences in HCV genotyping methods with regard to CRF 2k/1b and to gain insight in the prevalence of the CRF 2k/1b. Genotyping results by Versant HCV Genotype Assay were compared with nonstructural protein 5B (NS5B) sequencing. In total, from November 2001 until March 2015, 3296 serum samples were analyzed by Versant HCV Genotype Assay. As misclassified CRF is harbored among HCV genotype 2, we further focused our search on 142 (4.3%) samples positive for HCV genotype 2. On 116 (81.7%) retrieved samples, the NS5B sequencing was performed. Twelve out of the 116 retrieved samples (10.3%) were classified as CRF 2k/1b by sequencing of the NS5B region. Ten of these 12 samples were originally misclassified as genotype 2a or 2c, while 2 of them were misclassified as genotype 2. Our results show that the current prevalence of CRF 2k/1b is underestimated. The importance of correct HCV genotyping is emphasized, considering the tailored choice of treatment regimen and overall prognosis.

Rotavirus vaccine-derived cases in Belgium: Evidence for reversion of attenuating mutations and alternative causes of gastroenteritis.

Since the introduction of live-attenuated rotavirus vaccines in Belgium in 2006, surveillance has routinely detected rotavirus vaccine-derived strains. However, their genomic landscape and potential role in gastroenteritis have not been thoroughly investigated. We compared VP7 and VP4 nucleotide sequences obtained from rotavirus surveillance with the Rotarix vaccine sequence. As a result, we identified 80 vaccine-derived strains in 5125 rotavirus-positive infants with gastroenteritis from 2007 to 2018. Using both viral metagenomics and reverse transcription qPCR, we evaluated the vaccine strains and screened for co-infecting enteropathogens. Among the 45 patients with known vaccination status, 39 were vaccinated and 87% received the vaccine less than a month before the gastroenteritis episode. Reconstruction of 30 near complete vaccine-derived genomes revealed 0-11 mutations per genome, with 88% of them being non-synonymous. This, in combination with several shared amino acid changes among strains, pointed at selection of minor variant(s) present in the vaccine. We also found that some of these substitutions were true revertants (e.g., F167L on VP4, and I45T on NSP4). Finally, co-infections with known (e.g., Clostridioides difficile and norovirus) and divergent or emerging (e.g., human parechovirus A1, salivirus A2) pathogens were detected, and we estimated that 35% of the infants likely had gastroenteritis due to a 'non-rotavirus' cause. Conversely, we could not rule out the vaccine-derived gastroenteritis in over half of the cases. Continued studies inspecting reversion to pathogenicity should monitor the long-time safety of live-attenuated rotavirus vaccines. All in all, the complementary approach with NGS and qPCR provided a better understanding of rotavirus vaccine strain evolution in the Belgian population and epidemiology of co-infecting enteropathogens in suspected rotavirus vaccine-derived gastroenteritis cases.

Hepatitis E virus-associated haemophagocytic lymphohistiocytosis.

We report the first documented case of hepatitis E virus-associated haemophagocytic lymphohistiocytosis. This case emphasizes the fact that infectious agents other than those classically described should be taken into consideration as a potential trigger of virus-associated haemophagocytic syndrome. Prompt recognition is crucial to start early treatment of the underlying infection and possibly improve the outcome of this frequently fatal syndrome.

Macrolide resistance in Mycoplasma genitalium from female sex workers in Belgium.

OBJECTIVES: Mycoplasma genitalium is emerging as an aetiological agent of sexually transmitted infections (STIs). Although M. genitalium is commonly treated with azithromycin, macrolide resistance associated with point mutations in the 23S rRNA gene is emerging. METHODS: In this study, the prevalence of M. genitalium and macrolide resistance in female sex workers (FSW) in Belgium was evaluated by a prospective study conducted between 2015 and 2016. Vaginal swabs were sampled from 303 FSW who underwent testing for M. genitalium along with standard STI screening. All samples positive for M. genitalium were subsequently tested for mutations associated with macrolide resistance. RESULTS: M. genitalium was detected in 10.8% of participants and macrolide resistance-associated mutations (A2058G and A2059G) were found in 6.5% of isolates. CONCLUSIONS: M. genitalium is clearly present in FSW in Belgium. In contrast to other reports, for now the occurrence of macrolide resistance appears limited in this specific target population.

Performance evaluation of direct fluorescent antibody, Focus Diagnostics Simplexa™ Flu A/B & RSV and multi-parameter customized respiratory Taqman® array card in immunocompromised patients.

BACKGROUND: Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. OBJECTIVES: First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. STUDY DESIGN: We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. RESULTS: The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p<0.05). Performance characteristics of Simplexa™ testing were not significantly different compared to TAC testing (p>0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2€ and 2h for DFA, 31.8€ and 1.5h for Simplexa™ and 55€ and 3h for TAC testing. CONCLUSIONS: Performing the Simplexa™ test 24h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.

Diagnosis of hepatitis C virus genotype 2k/1b needs NS5B sequencing.

Hepatitis C virus (HCV) is probably the most common cause of liver cirrhosis and hepatocellular carcinoma worldwide. The correct identification of HCV genotype has important clinical implications as a marker of responsiveness to antiviral therapy and serves as a guideline for the duration of treatment. The VERSANT HCV Genotype 2.0 Assay failed to detect HCV genotype 2k/1b. HCV genotype 2k/1b detection requires NS5B sequencing.

Potential risk of misclassification HCV 2k/1b strains as HCV 2a/2c using VERSANT HCV Genotype 2.0 assay.

The performance of a hepatitis C virus (HCV) NS5B sequencing method described by Murphy et al. (Use of sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/E1 and 5' untranslated region sequences. J Clin Microbiol 2007;45(4):1102-12.) was compared with the VERSANT HCV Genotype 2.0 assay. The sequencing strategy led to detection of HCV recombinant genotype 2k/1b, previously identified as genotype 2a/2c, which reveals the importance of exact HCV genotyping and subtyping.

Clinical evaluation of a multi-parameter customized respiratory TaqMan(®) array card compared to conventional methods in immunocompromised patients.

BACKGROUND: Respiratory viral infections can cause significant morbidity and mortality in immunocompromised patients. Conventional tests routinely available at most institutions are limited by the number of detectable pathogens, by a poor sensitivity and/or a long turnaround time. OBJECTIVES: To compare the performance of routine conventional testing with direct fluorescent antibody assays and viral culture to a customized TaqMan® array card (TAC) real-time PCR method, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. STUDY DESIGN: We collected 143 respiratory samples from 120 symptomatic immunocompromised patients. Samples for which conventional and TAC results were discordant underwent further verification testing. RESULTS: The TAC assay identified viral pathogens in more samples than did conventional testing (77/143 versus 27/143; McNemar P<0.0001), even when TAC results for viruses that could not be detected by conventional testing were excluded from analysis (59/143 versus 26/143; P<0.0001). In addition, the TAC assay identified 18 samples with non-viral pathogens. Verification testing confirmed positive TAC results for 50 out of 55 samples for which conventional testing was negative. Two out of three samples with a positive conventional test but negative TAC result were confirmed positive. A viral and a total pathogen co-infection rate of 5.6% and 11.8% were found, respectively. CONCLUSIONS: The customized TAC assay resulted in a significantly increased identification of respiratory viruses. This study provides a practical real-life assessment of the performance of the TAC assay in a population for whom rapid and accurate diagnosis of viral and atypical pathogens is crucial for appropriate clinical management.