Use of a diagnostic Puumala virus real-time RT-PCR in an orthohantavirus endemic region in the Netherlands.

Laboratory diagnosis of orthohantavirus infection is primarily based on serology. However, for a confirmed serological diagnosis, evaluation of a follow-up serum sample is essential, which is time consuming and causes delay. Real-time reverse transcription polymerase chain reaction (RT-PCR) tests, if positive, provide an immediate and definitive diagnosis, and accurately identify the causative agent, where the discriminative nature of serology is suboptimal. We re-evaluated sera from orthohantavirus-suspected clinical cases in the Dutch regions of Twente and Achterhoek from July 2014 to April 2016 for the presence of Puumala orthohantavirus (PUUV), Tula orthohantavirus (TULV), and Seoul orthohantavirus (SEOV) RNA. PUUV RNA was detected in 11% of the total number (n = 85) of sera tested, in 50% of sera positive for anti-PUUV/TULV IgM (n = 16), and in 1.4% of sera negative or indeterminate for anti-PUUV/TULV IgM (n = 69). No evidence was found for the presence of TULV or SEOV viral RNA. Based on these findings, we propose two algorithms to implement real-time RT-PCR testing in routine orthohantavirus diagnostics, which optimally provide clinicians with early confirmed diagnoses and could prevent possible further invasive testing and treatment. IMPORTANCE: The addition of a real-time reverse transcription polymerase chain reaction test to routine orthohantavirus diagnostics may better aid clinical decision making than the use of standard serology tests alone. Awareness by clinicians and clinical microbiologists of this advantage may ultimately lead to a reduction in over-hospitalization and unnecessary invasive diagnostic procedures.

Ampicillin susceptibility testing of Haemophilus influenzae in the routine clinical laboratory by the EUCAST methodology compared to broth microdilution and the presence of ftsI gene mutations.

OBJECTIVES: To investigate the prevalence of ampicillin resistance in Haemophilus influenzae and the diagnostic accuracy of the EUCAST recommended disc diffusion method to detect the increasingly prevalent ampicillin resistance due to the presence of PBP3 alterations based on mutations in the ftsI gene. METHODS: During a 6-month period all consecutive non-duplicate H. influenzae isolates were prospectively collected and stored. MICs of ampicillin were determined by broth microdilution (BMD). PCR was performed to detect mutations in the ftsI gene. Results of routine disc diffusion susceptibility testing, including the penicillin screening test in accordance with the current EUCAST methodology, as well as additional Etest results, were compared to the BMD as the reference method. RESULTS: In 102 isolates, the prevalence of ampicillin resistance was 28% (29/102) by BMD. There was a good correlation between MICs of ampicillin and the presence of a ß-lactamase and/or an ftsI gene mutation. The prevalence of ampicillin resistance was overestimated using the EUCAST method (33% (34/102)) and underestimated when an additional Etest was used (24% (24/102)) (not significant). The sensitivity and specificity of the EUCAST methodology for the detection of ampicillin resistance were 97% ((28/29); 95% CI, 82-100%) and 92% ((67/73); 95% CI, 83-97%), respectively. CONCLUSIONS: The prevalence of ampicillin resistance was 28%, as determined by BMD. Although the overall diagnostic accuracy of the EUCAST ampicillin disc diffusion was high, misclassification of ampicillin susceptibility may still occur.

From a case-control survey to a diagnostic viral gastroenteritis panel for testing of general practitioners' patients.

OBJECTIVE: To evaluate the pathogenicity of a broad range of 11 possible gastroenteritis viruses, by means of statistical relationships with cases vs. controls, or Ct-values, in order to establish the most appropriate diagnostic panel for our general practitioner (GP) patients in the Netherlands (2010-2012). METHODS: Archived stool samples from 1340 cases and 1100 controls were retested using internally controlled multiplex real-time PCRs for putative pathogenic gastroenteritis viruses: adenovirus, astrovirus, bocavirus, enterovirus, norovirus GI and GII, human parechovirus, rotavirus, salivirus, sapovirus, and torovirus. RESULTS: The prevalence of any virus in symptomatic cases and asymptomatic controls was 16.6% (223/1340) and 10.2% (112/1100), respectively. Prevalence of astrovirus (adjusted odds ratio (aOR) 10.37; 95% confidence interval (CI) 1.34-80.06) and norovirus GII (aOR 3.10; CI 1.62-5.92) was significantly higher in cases versus controls. Rotavirus was encountered only in cases. We did not find torovirus and there was no statistically significant relationship with cases for salivirus (aOR 1,67; (CI) 0.43-6.54)), adenovirus non-group F (aOR 1.20; CI 0.75-1.91), bocavirus (aOR 0.85; CI 0.05-13.64), enterovirus (aOR 0.83; CI 0.50-1.37), human parechovirus (aOR 1.61; CI 0.54-4.77) and sapovirus (aOR 1.15; CI 0.67-1.98). Though adenovirus group F (aOR 6.37; CI 0.80-50.92) and norovirus GI (aOR 2.22, CI: 0.79-6.23) are known enteropathogenic viruses and were more prevalent in cases than in controls, this did not reach significance in this study. The Ct value did not discriminate between carriage and disease in PCR-positive subjects. CONCLUSIONS: In our population, diagnostic gastroenteritis tests should screen for adenovirus group F, astrovirus, noroviruses GI and GII, and rotavirus. Case-control studies as ours are lacking and should also be carried out in populations from other epidemiological backgrounds.

Correction: Bovine tuberculosis prevalence and risk factors in selected districts of Bangladesh.

[This corrects the article DOI: 10.1371/journal.pone.0241717.].

Zoonotic tuberculosis knowledge and practices among cattle handlers in selected districts of Bangladesh.

We assessed zoonotic tuberculosis (zTB) knowledge and prevention and control practices of 404 cattle handlers via a survey in three dairy-intensive districts of Bangladesh. Most respondents were aged 30-49 (52%) and male (95%). Almost all (99%) recognized the important public health burden of tuberculosis in Bangladesh, however, most (58%) had inadequate knowledge about zTB transmission to humans. Inappropriate practices such as: not using protective equipment (98%); smoking, drinking or eating food whilst working with cattle (69%); and sharing the same premises with animals (83%) were identified. Cattle handlers educated at secondary or higher levels were 2.82- (95% CI: 1.59-5.10) and 5.15 times (95% CI: 1.74-15.20) more likely to have adequate knowledge of control and prevention activities compared to those with no formal education. Those who had reared animals for 1-5 years were 2.67 times (95% CI: 1.44-4.91) more likely to have adequate knowledge, compared to those who reared animals for >15 years. Cattle handlers with a monthly incomes of 10,000-20,000 taka were significantly (Odds Ratio = 0.36, 95% CI: 0.14-0.92) less likely to have adequate knowledge compared to those with monthly incomes <10,000 taka. Cattle handlers with high school or higher education were 6.98 times (95% CI: 2.47-19.71) more likely to use appropriate zTB control and prevention practices compared to those without formal education. Those who had reared animals for 1-5 years, 6-10 years and 11-15 years were 2.72- (95% CI: 1.42-5.24), 2.49- (95% CI: 1.29-4.77) and 2.86 times (95% CI: 1.13-7.23) more likely to apply appropriate practices compared to those who reared animals for >15 years. Overall, education, duration of cattle rearing and monthly income predicted zTB knowledge and practices. There is an urgent need to educate those at high-risk of zTB transmission on issues including the handling of infected animals, and general hygiene. A One Health approach, to support the Sustainable Development Goals and the End TB strategy, appears to be the way forward.

Bovine tuberculosis prevalence and risk factors in selected districts of Bangladesh.

A cross-sectional survey was conducted in selected districts of Bangladesh to estimate the prevalence of bovine tuberculosis (bTB), and to identify the risk factors for bTB. We included 1865 farmed cattle from 79 herds randomly selected from five districts. Herd and animal level data were collected using semi-structured interviews with cattle herd owners. The single intradermal comparative tuberculin test (SICTT) was used to estimate the prevalence of bTB. The risk factors were identified using mixed-effect multiple logistic regression analyses. The overall herd and animal level prevalences of bTB were estimated to be 45.6% (95% Confidence Interval [CI] = 34.3-57.2%) and 11.3 (95% CI = 9.9-12.8%), respectively, using the OIE recommended >4 mm cut-off. The true animal level prevalence of bTB was estimated to be 11.8 (95% Credible Interval = 2.1-20.3%). At the herd level, farm size, bTB history of the farm and type of husbandry were significantly associated with bTB status in univariable analysis. Similarly, age group, sex, pregnancy status and parity were significantly associated with bTB at cattle level. However, in multivariable analysis only herd size at the herd level and age group and pregnancy status at the cattle level were significant. Compared to a herd size of 1-10, the odds of bTB were 22.8 (95% CI: 5.2-100.9) and 45.6 times (95% CI: 5.0-417.7) greater in herd sizes of >20-50 and >50, respectively. The odds of bTB were 2.2 (95% CI: 1.0-4.5) and 2.5 times (95% CI: 1.1-5.4) higher in cattle aged >3-6 years and > 6 years, compared to cattle aged ≤1 year. Pregnancy increased the odds of bTB by 1.7 times (95% CI: 1.2-2.4) compared to non-pregnant cattle. Taken together, the results suggest high herd and animal level prevalence of bTB in these 5 districts, with the greatest risk of bTB in older and pregnant cattle within large herds (>20), and highlight an urgent need for continued surveillance and implementation of bTB control programs in Bangladesh.

Retraction Note: Infection of great apes and a zoo keeper with the same Mycobacterium tuberculosis spoligotype.

The original article can be found online.

Distribution and relevance of Dientamoeba fragilis and Blastocystis species in gastroenteritis: results from a case-control study.

The actual role of Dientamoeba fragilis and Blastocystis in patients with gastrointestinal symptoms is still under debate. A multicenter case-control study was performed in The Netherlands to elucidate the clinical relevance of molecular diagnostics results in gastroenteritis (GE). Samples from this case-control study were used to perform a detailed analysis on the presence of D. fragilis and Blastocystis in relation to gastrointestinal symptoms. In the present study, a real-time PCR for Blastocystis was performed on 1374 case samples and 1026 control samples from the multicenter gastroenteritis case-control study previously tested for D. fragilis. Prevalence of both micro-organisms was highest in children under 20 years of age and lowest in the oldest age group. A significantly lower overall detection of D. fragilis and Blastocystis was found in cases (both 25.8%) as compared to controls (37.6% and 40.0%, respectively). The difference for D. fragilis was statistically significant for subjects above 20 years of age. For Blastocystis, the difference was statistically significant in all age groups, except in children less than 5 years of age. A negative relation between D. fragilis-positive cases and diarrhea was found in this study population. More GE symptoms were reported in cases without D. fragilis or Blastocystis. In the present study, prevalence of both D. fragilis and Blastocystis is lower in cases with gastroenteritic symptoms than in controls. Besides, in cases with D. fragilis or Blastocystis, no association is shown between any of the GE symptoms. Interestingly, this suggests that the presence of these protozoans may be considered characteristic of a healthy intestinal microbiome.

Selenite enrichment broth to improve the sensitivity in molecular diagnostics of Salmonella.

Selenite enrichment broth (SEB) is used to optimize the recovery of Salmonella enterica subspecies enterica from stool samples. Compared to a direct culture approach, it enhances culture yield by reducing growth of faecal coliforms and faecal streptococci. Over the course of seven years from 2000 to 2017, 47,235 faecal samples were tested with a Salmonella PCR. We investigated the added value of using SEB in combination with faeces for DNA extraction, in order to improve the sensitivity of molecular diagnostics for detection of Salmonella. A Salmonella enterica subspecies enterica strain was tested for growth characteristics, with and without incubation in SEB, to determine the impact of Selenite enrichment in the Salmonella PCR. Retrospectively, a total of 102 Salmonella enterica subspecies enterica PCR positive faecal samples were re-analysed. DNA extraction was performed with the EasyMag® and MagNaPure96® system using three different input volumes of faeces and SEB. Prospectively, 114 Salmonella PCR positive faecal samples were retested within 2 days using five different input volumes for DNA extraction. Retrospectively, PCR that used SEB as part of input in the DNA extraction, 7/102 (7%) Salmonella PCR positive samples were additionally detected compared to no use of SEB. Of these, Salmonella enterica subspecies enterica serovariation Thompson, Enteritidis, 9,12:l.v and Senftenberg have been outbreak related in the past. Prospectively results were combined in collaboration with another microbiology laboratory, 15/114 (13.2%) additional specimens were detected with the Salmonella PCR, including processing Selenite enrichment broth. In conclusion, of the total 47,235 feacal samples, with SEB the prevalence of a positive PCR for Salmonella is 2.2%. Of these 2.2% positive Salmonella PCRs, 0.4% was not detected in culture. By using SEB an improved detection of Salmonella diagnostics could be realized and a substantial part of 13,2% additional Salmonella cases could be detected.

Microbiological diagnosis of adult tuberculous meningitis in a ten-year cohort in Indonesia.

We evaluated microbiological diagnosis of tuberculous (TB) meningitis in a referral hospital in Indonesia. Over a ten-year period, we examined cerebrospinal fluid (CSF) samples of 1180 adult meningitis suspects. Sensitivity of different methods was compared, and results were stratified for HIV status, disease severity, and CSF volume. TB meningitis was bacteriologically confirmed in 501 patients. Using clinical diagnosis as reference standard (n = 713), sensitivity of different methods was 12.2% (86/703) for microscopy, 42% (73/174) for Xpert MTB/RIF, 46.0% (163/354) for solid culture, 48.8% (332/680) for liquid culture, and 64.0% (212/331) for in-house PCR. Head to head comparisons in 654 patients showed a higher yield of in-house PCR (32.3%) compared to culture (15.6%, P < 0.01). Microscopic observation of drug susceptibility (MODS) culture more rapidly became positive compared to other culture methods. Yield of culture was lower in HIV-infected (39/105) than in HIV-negative patients (N = 316/585; P < 0.01). Molecular and culture methods gave higher yields in patients with more severe disease (P < 0.01). CSF volume of ≥6 ml increased the yield of culture (42.8% versus 12.1% for CSF <6 ml, P < 0.01) and ZN-microscopy (18.3% versus 1.9% for CSF <6 ml, P < 0.01). CSF centrifugation had no clear effect on sensitivity of Xpert MTB/RIF. ZN-microscopy lacks sensitivity for diagnosis of TB meningitis. For molecular assays, in-house IS6110-PCR is more sensitive than Xpert MTB/RIF. MODS culture has a clear advantage in terms of speed. Large CSF volumes are necessary for all tests. The effect of CSF processing for Xpert MTB/RIF needs further study.

Correction: Variability and cost implications of three generations of the Roche LightCycler® 480.

[This corrects the article DOI: 10.1371/journal.pone.0190847.].

Variability and cost implications of three generations of the Roche LightCycler® 480.

Real time PCR has become a dominant method for the highly sensitive detection of pathogens in clinical material. Real time PCR can generate a fluorescence signal by using fluorescence labelled probes, allowing us to detect and semi quantify the amount of amplified DNA. Here we test the variability of the detection system and cost implications of three different versions of the LightCycler® 480 (LC480), focusing on the intensity of fluorescence and Cq in monoplex and multiplex rtPCRs. For gastro-intestinal pathogens there was no correlation between the intensity of fluorescence and the Cq value in the different LC480 types. For probes with the dyes FAMTM, HEXTM, Cy5 and Red610 a higher fluorescence intensity was seen in LC480 type II and III compared to LC480 type I. After lowering the probe concentration for the Cy5 dye three-fold (from 0.3µM to 0.1µM) the Cq value remains the same and the intensity of fluorescence decreases. For the LC480 type II and III the difference in fluorescence intensity was much more extreme. The concentration of the different labelled probes can be lowered at least six-fold in LC480 type II and III cyclers while maintaining a fluorescence intensity as high as achieved in the LC480 type I with undiluted probe. In conclusion, the strength of the fluorescence signal of the LightCycler® 480 type III is superior to that of LightCycler® 480 types I and II, allowing the use of lower probe concentrations for all dyes, particularly for the dyes Red610 and Cy5. This results in a two thirds reduction in PCR probe costs. Switching to these newer machines for real-time PCR can reduce dye labelled probe consumption and thus reduce costs significantly.

Genomic Characterization of Colistin Heteroresistance in Klebsiella pneumoniae during a Nosocomial Outbreak.

Klebsiella pneumoniae is emerging as an important nosocomial pathogen due to its rapidly increasing multidrug resistance, which has led to a renewed interest in polymyxin antibiotics, such as colistin, as antibiotics of last resort. However, heteroresistance (i.e., the presence of a subpopulation of resistant bacteria in an otherwise susceptible culture) may hamper the effectiveness of colistin treatment in patients. In a previous study, we showed that colistin resistance among extended-spectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates emerged after the introduction of selective digestive tract decontamination (SDD) in an intensive care unit (ICU). In this study, we investigated heteroresistance to colistin among ESBL-producing K. pneumoniae isolates by using population analysis profiles (PAPs). We used whole-genome sequencing (WGS) to identify the mutations that were associated with the emergence of colistin resistance in these K. pneumoniae isolates. We found five heteroresistant subpopulations, with colistin MICs ranging from 8 to 64 mg/liter, which were derived from five clonally related, colistin-susceptible clinical isolates. WGS revealed the presence of mutations in the lpxM, mgrB, phoQ, and yciM genes in colistin-resistant K. pneumoniae isolates. In two strains, mgrB was inactivated by an IS3-like or ISKpn14 insertion sequence element. Complementation in trans with the wild-type mgrB gene resulted in these strains reverting to colistin susceptibility. The MICs for colistin-susceptible strains increased 2- to 4-fold in the presence of the mutated phoQ, lpxM, and yciM alleles. In conclusion, the present study indicates that heteroresistant K. pneumoniae subpopulations may be selected for upon exposure to colistin. Mutations in mgrB and phoQ have previously been associated with colistin resistance, but we provide experimental evidence for roles of mutations in the yciM and lpxM genes in the emergence of colistin resistance in K. pneumoniae.

Paediatric nodal marginal zone B-cell lymphadenopathy of the neck: a Haemophilus influenzae-driven immune disorder?

Many hyperplasias and lymphomas of marginal zone B-cells are associated with infection. We identified six children and one adolescent with cervical lymphadenopathy showing prominent polyclonal nodal marginal zone hyperplasia (pNMZH) and four adolescents with monoclonal paediatric nodal marginal zone lymphoma (pNMZL). The clonality status was assessed using BIOMED-2-IG PCR analysis. Haemophilus influenzae was identified in all six cases of pNMZH that could be tested by direct culture (N = 3) or a very sensitive PCR for the H. influenzae gyrase gene in frozen materials (N = 5). H. influenzae was not detected in three pNMZLs and 28 non-specific reactive cervical lymph nodes of age-matched controls, except for a single control node that was obtained during oropharyngeal surgery for a cleft palate showing very low copy numbers of H. influenzae. pNMZH patients were younger than pNMZL patients (median age 12 versus 21 years). pNMZH showed a prominent nodular appearance with variable fibrosis without acute inflammation. Within the nodules, the expanded germinal centres and variably sized marginal zones were colonized by activated B-cells with weak expression of IgD and lack of CD10 and/or BCL6 expression. Some areas showed skewed light chain expression in plasma cells (4/5 cases lambda). In four cases tested, this was confirmed by flow cytometry for surface Ig (3/4 cases lambda). In contrast, pNMZL showed more extensive expansion of marginal zones by centrocytoid cells and often expression of BCL2 protein. Several H. influenzae strains are known to interact with the constant part of IgD on human B-cells, leading to their polyclonal proliferation and activation. We speculate that in vivo stimulation of IgD+ marginal zone B-cells by this bacterium may be implicated in this particular lymphadenopathy that should be distinguished from monoclonal pNMZL.

Optimization of standard in-house 24-locus variable-number tandem-repeat typing for Mycobacterium tuberculosis and its direct application to clinical material.

Variable-number tandem-repeat (VNTR) typing with a panel of 24 loci is the current gold standard in the molecular typing of Mycobacterium tuberculosis complex isolates. However, because of technical problems, a part of the loci often cannot be amplified by multiplex PCRs. Therefore, a considerable number of single-locus PCRs have to be performed for the loci with missing results, which impairs the laboratory work flow. Therefore, the original in-house method described by Supply et al. in 2006 was reevaluated. We modified seven primers and the PCR master mixture and obtained a strongly optimized in-house 24-locus VNTR typing method. The percentage of instantly complete 24-locus VNTR patterns detected in the routine flow of typing activities increased to 84.7% from the 72.3% obtained with the typing conducted with the commercially available Genoscreen MIRU-VNTR typing kit. The analytical sensitivity of the optimized in-house method was assessed by serial dilutions of M. tuberculosis in bronchoalveolar lavage fluid. A 1:10 dilution of the different strains tested was the lowest dilution for the detection of a complete 24-locus VNTR pattern. The optimized in-house 24-locus VNTR typing method will reduce the turnaround time of typing significantly and also the financial burden of these activities.

Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC.

BACKGROUND: Resistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC. METHODS: A real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates. RESULTS: Both sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons. CONCLUSIONS: In this study, we present a multiplex real-time PCR that is a robust, reliable and rapid method for the detection of the most prevalent carbapenemases blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC, and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures.

Infection of great apes and a zoo keeper with the same Mycobacterium tuberculosis spoligotype.

An animal keeper was diagnosed with pulmonary tuberculosis (TB) after bi-annual screening for latent TB infection in zoo employees. In the same period, several bonobos of the zoo were suffering from TB as well. The Mycobacterium tuberculosis strains from both the animal keeper and the bonobos appeared identical. We provide evidence that the animals infected their keeper.

Evaluation of yield of currently available diagnostics by sample type to optimize detection of respiratory pathogens in patients with a community-acquired pneumonia.

BACKGROUND: For the detection of respiratory pathogens, the sampling strategy may influence the diagnostic yield. Ideally, samples from the lower respiratory tract are collected, but they are difficult to obtain. OBJECTIVES: In this study, we compared the diagnostic yield in sputum and oropharyngeal samples (OPS) for the detection of respiratory pathogens in patients with community-acquired pneumonia (CAP), with the objective to optimize our diagnostic testing algorithm. METHODS: Matched sputum samples, OPS, blood cultures, serum, and urine samples were taken from patients (>18 years) with CAP and tested for the presence of possible respiratory pathogens using bacterial cultures, PCR for 17 viruses and five bacteria and urinary antigen testing. RESULTS: When using only conventional methods, that is, blood cultures, sputum culture, urinary antigen tests, a pathogen was detected in 49·6% of patients (n = 57). Adding molecular detection assays increased the yield to 80%. A pathogen was detected in 77 of the 115 patients in OPS or sputum samples by PCR. The sensitivity of the OPS was lower than that of the sputum samples (57% versus 74%). In particular, bacterial pathogens were more often detected in sputum samples. The sensitivity of OPS for the detection of most viruses was higher than in sputum samples (72% versus 66%), except for human rhinovirus and respiratory syncytial virus. CONCLUSION: Addition of PCR on both OPS and sputum samples significantly increased the diagnostic yield. For molecular detection of bacterial pathogens, a sputum sample is imperative, but for detection of most viral pathogens, an OPS is sufficient.