, a cluster of cases of pneumonia of unknown etiology were reported linked to a market in Wuhan, China 1. The causative agent was identified as the species Severe acute respiratory syndrome-related coronavirus and was named SARS-CoV-2 (ref. 2). By 16 April the virus had spread to 185 different countries, infected over 2,000,000 people and resulted in over 130,000 deaths 3. In the Netherlands, the first case of SARS-CoV-2 was notified on 27 February. The outbreak started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also outside, the south of the Netherlands. The combination of near to real-time whole-genome sequence analysis and epidemiology resulted in reliable assessments of the extent of SARS-CoV-2 transmission in the community, facilitating early decision-making to control local transmission of SARS-CoV-2 in the Netherlands. We demonstrate how these data were generated and analyzed, and how SARS-CoV-2 whole-genome sequencing, in combination with epidemiological data, was used to inform public health decision-making in the Netherlands. Whole-genome sequencing (WGS) is a powerful tool to understand the transmission dynamics of outbreaks and inform outbreak control decisions 4-7. Evidence of this was seen during the 2014-2016 West African Ebola outbreak when real-time WGS was used to help public health decision-making, a strategy dubbed 'precision public health pathogen genomics' 8,9. Immediate sharing and analysis of data during outbreaks is now recommended as an integral part of outbreak response 10-12. Feasibility of real-time WGS requires access to sequence platforms that provide reliable sequences, access to metadata for interpretation, and data analysis at high speed and low cost. Therefore, WGS for outbreak support is an active area of research. Nanopore sequencing has been employed in recent outbreaks of Usutu, Ebola, Zika and yellow fever virus owing to the ease of use and relatively low start-up cost 4-7. The robustness of this method has recently been validated using Usutu virus 13,14. In the Netherlands, the first COVID-19 case was confirmed on 27 February and WGS was performed in near to real-time using an amplicon-based sequencing approach. From 22 January, symptomatic travelers from countries where SARS-CoV-2 was known to circulate were routinely tested. The first case of SARS-CoV-2 infection in the Netherlands was identified on 27 February in a person with recent travel history to Italy and an additional case was identified one day later, also in a person with recent travel history to Italy. The genomes of these first two positive samples were generated and analyzed by 29 February. These two viruses clustered differently in the phylogenetic tree, confirming separate introductions (Fig. 1a). The advice to test hospitalized patients with serious respiratory infections was issued on 24 February and subsequent attempts to identify possible local transmission chains triggered testing for SARS-CoV-2 on a large scale in h...

Tropical infectious diseases diagnosis and surveillance are often hampered by difficulties of sample collection and transportation. Filter paper potentially provides a useful medium to help overcome such problems. We reviewed the literature on the use of filter paper, focusing on the evaluation of nucleic acid and serological assays for diagnosis of infectious diseases using dried blood spots (DBS) compared with recognized gold standards. We reviewed 296 eligible studies and included 101 studies evaluating DBS and 192 studies on other aspects of filter paper use. We also discuss the use of filter paper with other body fluids and for tropical veterinary medicine. In general, DBS perform with sensitivities and specificities similar or only slightly inferior to gold standard sample types. However, important problems were revealed with the uncritical use of DBS, inappropriate statistical analysis, and lack of standardized methodology. DBS have great potential to empower healthcare workers by making laboratory-based diagnostic tests more readily accessible, but additional and more rigorous research is needed.

BackgroundDried blood spots (DBS) have been used as alternative specimens to plasma to increase access to HIV viral load (VL) monitoring and early infant diagnosis (EID) in remote settings. We systematically reviewed evidence on the performance of DBS compared to plasma for VL monitoring and EID.Methods and FindingsThirteen peer reviewed HIV VL publications and five HIV EID papers were included. Depending on the technology and the viral load distribution in the study population, the percentage of DBS samples that are within 0.5 log of VL in plasma ranged from 52–100%. Because the input sample volume is much smaller in a blood spot, there is a risk of false negatives with DBS. Sensitivity of DBS VL was found to be 78–100% compared to plasma at VL below 1000 copies/ml, but this increased to 100% at a threshold of 5000 copies/ml. Unlike a plasma VL test which measures only cell free HIV RNA, a DBS VL also measures proviral DNA as well as cell-associated RNA, potentially leading to false positive results when using DBS. The systematic review showed that specificity was close to 100% at DBS VL above 5000 copies/ml, and this threshold would be the most reliable for predicting true virologic failure using DBS. For early infant diagnosis, DBS has a sensitivity of 100% compared to fresh whole blood or plasma in all studies.ConclusionsAlthough limited data are available for EID, DBS offer a highly sensitive and specific sampling strategy to make viral load monitoring and early infant diagnosis more accessible in remote settings. A standardized approach for sampling, storing, and processing DBS samples would be essential to allow successful implementation.Trial RegistrationPROSPERO Registration #: CRD42013003621.

Clinical studies of 6- and 12-valent pneumococcal capsular polysaccharide vaccines were carried out in controlled studies among novice gold miners in South Africa. In the studies 1,523 persons received pneumococcal vaccine, and 3,171 were included as controls. In the great majority of subjects given pneumococcal vaccine, antibodies developed against the capsular types included in the vaccine. The 6-valent vaccine afforded 76% reduction in cases of laboratory-verified pneumococcal pneumonia caused by the homologous types, and there was 92% reduction in the cases afforded by the 12-valent vaccine. The vaccines were well tolerated and offer great promise for effective control of illnesses caused by pneumococci.

SummaryBackgroundIn southeast Asia, antibiotic prescription in febrile patients attending primary care is common, and a probable contributor to the high burden of antimicrobial resistance. The objective of this trial was to explore whether C-reactive protein (CRP) testing at point of care could rationalise antibiotic prescription in primary care, comparing two proposed thresholds to classify CRP concentrations as low or high to guide antibiotic treatment.MethodsWe did a multicentre, open-label, randomised, controlled trial in participants aged at least 1 year with a documented fever or a chief complaint of fever (regardless of previous antibiotic intake and comorbidities other than malignancies) recruited from six public primary care units in Thailand and three primary care clinics and one outpatient department in Myanmar. Individuals were randomly assigned using a computer-based randomisation system at a ratio of 1:1:1 to either the control group or one of two CRP testing groups, which used thresholds of 20 mg/L (group A) or 40 mg/L CRP (group B) to guide antibiotic prescription. Health-care providers were masked to allocation between the two intervention groups but not to the control group. The primary outcome was the prescription of any antibiotic from day 0 to day 5 and the proportion of patients who were prescribed an antibiotic when CRP concentrations were above and below the 20 mg/L or 40 mg/L thresholds. The primary outcome was analysed in the intention-to-treat and per-protocol populations. The trial is registered with ClinicalTrials.gov, number NCT02758821, and is now completed.FindingsBetween June 8, 2016, and Aug 25, 2017, we recruited 2410 patients, of whom 803 patients were randomly assigned to CRP group A, 800 to CRP group B, and 807 to the control group. 598 patients in CRP group A, 593 in CRP group B, and 767 in the control group had follow-up data for both day 5 and day 14 and had been prescribed antibiotics (or not) in accordance with test results (per-protocol population). During the trial, 318 (39%) of 807 patients in the control group were prescribed an antibiotic by day 5, compared with 290 (36%) of 803 patients in CRP group A and 275 (34%) of 800 in CRP group B. The adjusted odds ratio (aOR) of 0·80 (95% CI 0·65–0·98) and risk difference of −5·0 percentage points (95% CI −9·7 to −0·3) between group B and the control group were significant, although lower than anticipated, whereas the reduction in prescribing in group A compared with the control group was not significant (aOR 0·86 [0·70–1·06]; risk difference −3·3 percentage points [–8·0 to 1·4]). Patients with high CRP concentrations in both intervention groups were more likely to be prescribed an antibiotic than in the control group (CRP ≥20 mg/L: group A vs control group, p<0·0001; CRP ≥40 mg/L: group B vs control group, p<0·0001), and those with low CRP concentrations were more likely to have an antibiotic withheld (CRP <20 mg/L: group A vs control group, p<0·0001; CRP <40 mg/L: group B vs control group, p<0·0001). 24 serious adverse eve...

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BackgroundViral load (VL) monitoring is the standard of care in developing country settings for detecting HIV treatment failure. Since 2010 the World Health Organization has recommended a phase-in approach to VL monitoring in resource-limited settings. We conducted a systematic review of the accuracy and precision of HIV VL technologies for treatment monitoring.Methods and FindingsA search of Medline and Embase was conducted for studies evaluating the accuracy or reproducibility of commercially available HIV VL assays. 37 studies were included for review including evaluations of the Amplicor Monitor HIV-1 v1.5 (n = 25), Cobas TaqMan v2.0 (n = 11), Abbott RealTime HIV-1 (n = 23), Versant HIV-1 RNA bDNA 3.0 (n = 15), Versant HIV-1 RNA kPCR 1.0 (n = 2), ExaVir Load v3 (n = 2), and NucliSens EasyQ v2.0 (n = 1). All currently available HIV VL assays are of sufficient sensitivity to detect plasma virus levels at a lower detection limit of 1,000 copies/mL. Bias data comparing the Abbott RealTime HIV-1, TaqMan v2.0 to the Amplicor Monitor v1.5 showed a tendency of the Abbott RealTime HIV-1 to under-estimate results while the TaqMan v2.0 overestimated VL counts. Compared to the Amplicor Monitor v1.5, 2–26% and 9–70% of results from the Versant bDNA 3.0 and Abbott RealTime HIV-1 differed by greater than 0.5log10. The average intra and inter-assay variation of the Abbott RealTime HIV-1 were 2.95% (range 2.0–5.1%) and 5.44% (range 1.17–30.00%) across the range of VL counts (2log10–7log10).ConclusionsThis review found that all currently available HIV VL assays are of sufficient sensitivity to detect plasma VL of 1,000 copies/mL as a threshold to initiate investigations of treatment adherence or possible treatment failure. Sources of variability between VL assays include differences in technology platform, plasma input volume, and ability to detect HIV-1 subtypes. Monitoring of individual patients should be performed on the same technology platform to ensure appropriate interpretation of changes in VL.Prospero registration # CD42013003603.

Vaginal lactobacilli assessed by PCR-based microarray and PCR-based genotyping of HPV in South African women at risk for HIV and BV. Vaginal lactobacilli can be defined by microarray techniques in fixed cervical samples of South African women. Cervical brush samples suspended in the coagulant fixative BoonFix of one hundred women attending a health centre for HIV testing in South Africa were available for this study. In the Ndlovu Medical Centre in Elandsdoorn, South Africa, identification of 18 hr-HPV genotypes was done using the INNO-LiPA method. An inventory of lactobacilli organisms was performed using microarray technology. On the basis of the Lactobacillus and Lactobacillus biofilm scoring, the cases were identified as Leiden bacterial vaginosis (BV) negative (BV-; n = 41), Leiden BV intermediate (BV±; n = 25), and Leiden BV positive (BV+; n = 34). Fifty-one women were HIV positive and 49 HIV negative. Out of the 51 HIV positive women, 35 were HPV infected. These 51 HIV positive women were frequently infected with HPV16 and HPV18. In addition, HPV35, HPV52, HPV33, and HPV66 were often detected in these samples. Lactobacillus salivarius and Lactobacillus iners were the most prevalent lactobacilli as established by the microarray technique. In women with HPV infection, the prevalence of Lactobacillus crispatus was significantly reduced. In both HIV and HPV infection, a similar (but not identical) shift in the composition of the lactobacillus flora was observed. We conclude that there is a shift in the composition of vaginal lactobacilli in HIV-infected women. Because of the prominence of HPV35, HPV52, HPV33, and HPV66, vaccination for exclusively HPV16 and HPV18 might be insufficient in South African HIV+ women.