Routine Metagenomics Service for ICU Patients with Respiratory Infection.

Rationale: Respiratory metagenomics (RMg) needs evaluation in a pilot service setting to determine utility and inform implementation into routine clinical practice. Objectives: Feasibility, performance, and clinical impacts on antimicrobial prescribing and infection control were recorded during a pilot RMg service. Methods: RMg was performed on 128 samples from 87 patients with suspected lower respiratory tract infection (LRTI) on two general and one specialist respiratory ICUs at Guy's and St Thomas' NHS Foundation Trust, London. Measurements and Main Results: During the first 15 weeks, RMg provided same-day results for 110 samples (86%), with a median turnaround time of 6.7 hours (interquartile range = 6.1-7.5 h). RMg was 93% sensitive and 81% specific for clinically relevant pathogens compared with routine testing. Forty-eight percent of RMg results informed antimicrobial prescribing changes (22% escalation; 26% deescalation) with escalation based on speciation in 20 out of 24 cases and detection of acquired-resistance genes in 4 out of 24 cases. Fastidious or unexpected organisms were reported in 21 samples, including anaerobes (n = 12), Mycobacterium tuberculosis, Tropheryma whipplei, cytomegalovirus, and Legionella pneumophila ST1326, which was subsequently isolated from the bedside water outlet. Application to consecutive severe community-acquired LRTI cases identified Staphylococcus aureus (two with SCCmec and three with luk F/S virulence determinants), Streptococcus pyogenes (emm1-M1uk clone), S. dysgalactiae subspecies equisimilis (STG62647A), and Aspergillus fumigatus with multiple treatments and public health impacts. Conclusions: This pilot study illustrates the potential of RMg testing to provide benefits for antimicrobial treatment, infection control, and public health when provided in a real-world critical care setting. Multicenter studies are now required to inform future translation into routine service.

Dynamics of Salmonella enterica and antimicrobial resistance in the Brazilian poultry industry and global impacts on public health.

Non-typhoidal Salmonella enterica is a common cause of diarrhoeal disease; in humans, consumption of contaminated poultry meat is believed to be a major source. Brazil is the world's largest exporter of chicken meat globally, and previous studies have indicated the introduction of Salmonella serovars through imported food products from Brazil. Here we provide an in-depth genomic characterisation and evolutionary analysis to investigate the most prevalent serovars and antimicrobial resistance (AMR) in Brazilian chickens and assess the impact to public health of products contaminated with S. enterica imported into the United Kingdom from Brazil. To do so, we examine 183 Salmonella genomes from chickens in Brazil and 357 genomes from humans, domestic poultry and imported Brazilian poultry products isolated in the United Kingdom. S. enterica serovars Heidelberg and Minnesota were the most prevalent serovars in Brazil and in meat products imported from Brazil into the UK. We extended our analysis to include 1,259 publicly available Salmonella Heidelberg and Salmonella Minnesota genomes for context. The Brazil genomes form clades distinct from global isolates, with temporal analysis suggesting emergence of these Salmonella Heidelberg and Salmonella Minnesota clades in the early 2000s, around the time of the 2003 introduction of the Enteritidis vaccine in Brazilian poultry. Analysis showed genomes within the Salmonella Heidelberg and Salmonella Minnesota clades shared resistance to sulphonamides, tetracyclines and beta-lactams conferred by sul2, tetA and blaCMY-2 genes, not widely observed in other co-circulating serovars despite similar selection pressures. The sul2 and tetA genes were concomitantly carried on IncC plasmids, whereas blaCMY-2 was either co-located with the sul2 and tetA genes on IncC plasmids or independently on IncI1 plasmids. Long-term surveillance data collected in the UK showed no increase in the incidence of Salmonella Heidelberg or Salmonella Minnesota in human cases of clinical disease in the UK following the increase of these two serovars in Brazilian poultry. In addition, almost all of the small number of UK-derived genomes which cluster with the Brazilian poultry-derived sequences could either be attributed to human cases with a recent history of foreign travel or were from imported Brazilian food products. These findings indicate that even should Salmonella from imported Brazilian poultry products reach UK consumers, they are very unlikely to be causing disease. No evidence of the Brazilian strains of Salmonella Heidelberg or Salmonella Minnesota were observed in UK domestic chickens. These findings suggest that introduction of the Salmonella Enteritidis vaccine, in addition to increasing antimicrobial use, could have resulted in replacement of salmonellae in Brazilian poultry flocks with serovars that are more drug resistant, but less associated with disease in humans in the UK. The plasmids conferring resistance to beta-lactams, sulphonamides and tetracyclines likely conferred a competitive advantage to the Salmonella Minnesota and Salmonella Heidelberg serovars in this setting of high antimicrobial use, but the apparent lack of transfer to other serovars present in the same setting suggests barriers to horizontal gene transfer that could be exploited in intervention strategies to reduce AMR. The insights obtained reinforce the importance of One Health genomic surveillance.

Determination and quantification of microbial communities and antimicrobial resistance on food through host DNA-depleted metagenomics.

Food products carry bacteria unless specifically sterilised. These bacteria can be pathogenic, commensal or associated with food spoilage, and may also be resistant to antimicrobials. Current methods for detecting bacteria on food rely on culturing for specific bacteria, a time-consuming process, or 16S rRNA metabarcoding that can identify different taxa but not their genetic content. Directly sequencing metagenomes of food is inefficient as its own DNA vastly outnumbers the bacterial DNA present. We optimised host DNA depletion enabling efficient sequencing of food microbiota, thereby increasing the proportion of non-host DNA sequenced 13-fold (mean; range: 1.3-40-fold) compared to untreated samples. The method performed best on chicken, pork and leafy green samples which had high mean prokaryotic read proportions post-depletion (0.64, 0.74 and 0.74, respectively), with lower mean prokaryotic read proportions in salmon (0.50) and prawn samples (0.19). We show that bacterial compositions and concentrations of antimicrobial resistance (AMR) genes differed by food type, and that salmon metagenomes were influenced by the production/harvesting method. The approach described in this study is an efficient and effective method of identifying and quantifying the predominant bacteria and AMR genes on food.

Multicentre evaluation of two multiplex PCR platforms for the rapid microbiological investigation of nosocomial pneumonia in UK ICUs: the INHALE WP1 study.

BACKGROUND: Culture-based microbiological investigation of hospital-acquired or ventilator-associated pneumonia (HAP or VAP) is insensitive, with aetiological agents often unidentified. This can lead to excess antimicrobial treatment of patients with susceptible pathogens, while those with resistant bacteria are treated inadequately for prolonged periods. Using PCR to seek pathogens and their resistance genes directly from clinical samples may improve therapy and stewardship. METHODS: Surplus routine lower respiratory tract samples were collected from intensive care unit patients about to receive new or changed antibiotics for hospital-onset lower respiratory tract infections at 15 UK hospitals. Testing was performed using the BioFire FilmArray Pneumonia Panel (bioMérieux) and Unyvero Pneumonia Panel (Curetis). Concordance analysis compared machine and routine microbiology results, while Bayesian latent class (BLC) analysis estimated the sensitivity and specificity of each test, incorporating information from both PCR panels and routine microbiology. FINDINGS: In 652 eligible samples; PCR identified pathogens in considerably more samples compared with routine microbiology: 60.4% and 74.2% for Unyvero and FilmArray respectively vs 44.2% by routine microbiology. PCR tests also detected more pathogens per sample than routine microbiology. For common HAP/VAP pathogens, FilmArray had sensitivity of 91.7%-100.0% and specificity of 87.5%-99.5%; Unyvero had sensitivity of 50.0%-100.0%%, and specificity of 89.4%-99.0%. BLC analysis indicated that, compared with PCR, routine microbiology had low sensitivity, ranging from 27.0% to 69.4%. INTERPRETATION: Conventional and BLC analysis demonstrated that both platforms performed similarly and were considerably more sensitive than routine microbiology, detecting potential pathogens in patient samples reported as culture negative. The increased sensitivity of detection realised by PCR offers potential for improved antimicrobial prescribing.

Clinical Metagenomic Sequencing for Species Identification and Antimicrobial Resistance Prediction in Orthopedic Device Infection.

Diagnosis of orthopedic device-related infection is challenging, and causative pathogens may be difficult to culture. Metagenomic sequencing can diagnose infections without culture, but attempts to detect antimicrobial resistance (AMR) determinants using metagenomic data have been less successful. Human DNA depletion may maximize the amount of microbial DNA sequence data available for analysis. Human DNA depletion by saponin was tested in 115 sonication fluid samples generated following revision arthroplasty surgery, comprising 67 where pathogens were detected by culture and 48 culture-negative samples. Metagenomic sequencing was performed on the Oxford Nanopore Technologies GridION platform. Filtering thresholds for detection of true species versus contamination or taxonomic misclassification were determined. Mobile and chromosomal genetic AMR determinants were identified in Staphylococcus aureus-positive samples. Of 114 samples generating sequence data, species-level positive percent agreement between metagenomic sequencing and culture was 50/65 (77%; 95% confidence interval [CI], 65 to 86%) and negative percent agreement was 103/114 (90%; 95% CI, 83 to 95%). Saponin treatment reduced the proportion of human bases sequenced in comparison to 5-µm filtration from a median (interquartile range [IQR]) of 98.1% (87.0% to 99.9%) to 11.9% (0.4% to 67.0%), improving reference genome coverage at a 10-fold depth from 18.7% (0.30% to 85.7%) to 84.3% (12.9% to 93.8%). Metagenomic sequencing predicted 13/15 (87%) resistant and 74/74 (100%) susceptible phenotypes where sufficient data were available for analysis. Metagenomic nanopore sequencing coupled with human DNA depletion has the potential to detect AMR in addition to species detection in orthopedic device-related infection. Further work is required to develop pathogen-agnostic human DNA depletion methods, improving AMR determinant detection and allowing its application to other infection types.

An optimised protocol for detection of SARS-CoV-2 in stool.

BACKGROUND: SARS-CoV-2 has been detected in stool samples of COVID-19 patients, with potential implications for faecal-oral transmission. Compared to nasopharyngeal swab samples, the complexity of the stool matrix poses a challenge in the detection of the virus that has not yet been solved. However, robust and reliable methods are needed to estimate the prevalence and persistence of SARS-CoV-2 in the gut and to ensure the safety of microbiome-based procedures such as faecal microbiota transplant (FMT). The aim of this study was to establish a sensitive and reliable method for detecting SARS-CoV-2 in stool samples. RESULTS: Stool samples from individuals free of SARS-CoV-2 were homogenised in saline buffer and spiked with a known titre of inactivated virus ranging from 50 to 750 viral particles per 100 mg stool. Viral particles were concentrated by ultrafiltration, RNA was extracted, and SARS-CoV-2 was detected via real-time reverse-transcription polymerase chain reaction (RT-qPCR) using the CDC primers and probes. The RNA extraction procedure we used allowed for the detection of SARS-CoV-2 via RT-qPCR in most of the stool samples tested. We could detect as few as 50 viral particles per 100 mg of stool. However, high variability was observed across samples at low viral titres. The primer set targeting the N1 region provided more reliable and precise results and for this primer set our method had a limit of detection of 1 viral particle per mg of stool. CONCLUSIONS: Here we describe a sensitive method for detecting SARS-CoV-2 in stool samples. This method can be used to establish the persistence of SARS-CoV-2 in stool and ensure the safety of clinical practices such as FMT.

Optimizing DNA Extraction Methods for Nanopore Sequencing of Neisseria gonorrhoeae Directly from Urine Samples.

Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient Neisseria gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae-spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated Chlamydia coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.

Metagenomic identification of severe pneumonia pathogens in mechanically-ventilated patients: a feasibility and clinical validity study.

BACKGROUND: Metagenomic sequencing of respiratory microbial communities for pathogen identification in pneumonia may help overcome the limitations of culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore™ sequencing of clinical respiratory specimens. METHODS: We conducted a case-control study of mechanically-ventilated patients with pneumonia (nine culture-positive and five culture-negative) and without pneumonia (eight controls). We collected endotracheal aspirates and applied a microbial DNA enrichment method prior to metagenomic sequencing with the Oxford Nanopore MinION device. For reference, we compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing. RESULTS: Human DNA depletion enabled in depth sequencing of microbial communities. In culture-positive cases, Nanopore revealed communities with high abundance of the bacterial or fungal species isolated by cultures. In four cases with resistant clinical isolates, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance in antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and Candida colonization in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa. CONCLUSIONS: We demonstrated technical feasibility and proof-of-concept clinical validity of Nanopore metagenomics for severe pneumonia diagnosis, with striking concordance with positive microbiologic cultures, and clinically actionable information obtained from sequencing in culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes.

Performance of urine lipoarabinomannan assays for paediatric tuberculosis in Tanzania.

We evaluated the diagnostic performance of two tests based on the release of lipoarabinomannan (LAM) into the urine, the MTB-LAM-ELISA assay and the Determine TB-LAM-strip assay, in children with suspected tuberculosis (TB) in a high TB/HIV-prevalence setting.In a prospective study, 132 children with suspected active TB were assigned to diagnostic subgroups. Urine samples were subjected to testing by both assays to ascertain sensitivity and specificity. Host factors associated with positive LAM results were investigated and LAM excretion monitored after antituberculous treatment initiation.18 (13.6%) children had culture-confirmed pulmonary TB. The assays' sensitivity was higher in HIV-positive versus HIV-negative children: 70% (95% confidence interval 35-93%) versus 13% (0-53%) for MTB-LAM-ELISA and 50% (19-81%) versus 0% (0-37%) for Determine TB-LAM. In 35 (27%) children with excluded active TB, both assays showed a specificity of 97.1% (85-100%). Proteinuria and low body mass index were independently associated with LAM positivity. In most patients, LAM excretion declined to zero during or at conclusion of antituberculous treatment.HIV/TB co-infected children might benefit from LAM-based tests to aid early TB diagnosis and subsequent positive impact on morbidity and mortality. Using LAM as a rule-in and treatment-monitoring tool may also show further potential.

Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection.

BACKGROUND: The timely and accurate diagnosis of bloodstream infection (BSI) is critical for patient management. With longstanding challenges for routine blood culture, metagenomics is a promising approach to rapidly provide sequence-based detection and characterisation of bloodborne bacteria. Long-read sequencing technologies have successfully supported the use of clinical metagenomics for syndromes such as respiratory illness, and modified approaches may address two requisite factors for metagenomics to be used as a BSI diagnostic: depletion of the high level of host DNA to then detect the low abundance of microbes in blood. METHODS: Blood samples from healthy donors were spiked with different concentrations of four prevalent causative species of BSI. All samples were then subjected to a modified saponin-based host DNA depletion protocol and optimised DNA extraction, whole genome amplification and debranching steps in preparation for sequencing, followed by bioinformatical analyses. Two related variants of the protocol are presented: 1mL of blood processed without bacterial enrichment, and 5mL of blood processed following a rapid bacterial enrichment protocol-SepsiPURE. RESULTS: After first identifying that a large proportion of host mitochondrial DNA remained, the host depletion process was optimised by increasing saponin concentration to 3% and scaling the reaction to allow more sample volume. Compared to non-depleted controls, the 3% saponin-based depletion protocol reduced the presence of host chromosomal and mitochondrial DNA < 106 and < 103 fold respectively. When the modified depletion method was further combined with a rapid bacterial enrichment method (SepsiPURE; with 5mL blood samples) the depletion of mitochondrial DNA improved by a further > 10X while also increasing detectable bacteria by > 10X. Parameters during DNA extraction, whole genome amplification and long-read sequencing were also adjusted, and subsequently amplicons were detected for each input bacterial species at each of the spiked concentrations, ranging from 50-100 colony forming units (CFU)/mL to 1-5 CFU/mL. CONCLUSION: In this proof-of-concept study, four prevalent BSI causative species were detected in under 12 h to species level (with antimicrobial resistance determinants) at concentrations relevant to clinical blood samples. The use of a rapid and precise metagenomic protocols has the potential to advance the diagnosis of BSI.

Use of the Xpert(®) MTB/RIF assay for diagnosing pulmonary tuberculosis comorbidity and multidrug-resistant TB in obstetrics and gynaecology inpatient wards at the University Teaching Hospital, Lusaka, Zambia.

OBJECTIVES: In high-tuberculosis (TB)-endemic countries, comorbidity of pulmonary TB in hospitalised patients with non-communicable diseases is well documented. In this study, we evaluated the use of the Xpert(®) MTB/RIF assay for the detection of concomitant pulmonary TB in patients admitted to the University Teaching Hospital, Lusaka, Zambia, with a primary obstetric or gynaecological condition. METHODS: The Study population were inpatients admitted with a primary obstetric or gynaecological problem who had a concomitant cough and were able to expectorate a sputum sample. Sputum samples from 94 patients were analysed for the presence of Mycobacterium tuberculosis (M.tb) by standard smear microscopy, MGIT culture, MGIT drug-susceptibility testing (DST) and the Xpert(®) MTB/RIF assay. The sensitivity and specificity of the Xpert(®) MTB/RIF assay were evaluated against the culture gold standard. RESULTS: Twenty-six of 94 (27.7%) patients had culture-confirmed pulmonary TB. The Xpert(®) MTB/RIF assay had a sensitivity of 80.8% [95% CI: 60.0-92.7%]) compared against MGIT culture. The Xpert(®) MTB/RIF assay was more sensitive than sputum smear microscopy (21/26 (80.8%) vs. 13/26 (50.0%), P = 0.02) and detected an additional eight culture-confirmed cases. Culture DST analysis identified two monoresistant M.tb strains: one resistant to rifampicin (rifampicin sensitive by the Xpert(®) MTB/RIF assay) and one to ethambutol. HIV infection was linked with a 3-fold increase in risk of TB, accounting for 87.5% (21/24) of TB cases. 50% of cases presented as comorbidities with other communicable diseases (CDs) and non-communicable diseases (NCDs). CONCLUSIONS: As an alternative to sputum microscopy, the Xpert(®) MTB/RIF assay provides a sensitive, specific and rapid method for the diagnosis of pulmonary TB in obstetric or gynaecological inpatients. Pulmonary TB is an important cause of concomitant comorbidity to the obstetric or gynaecological condition necessitating admission. TB and HIV comorbidities with other communicable and non-communicable diseases were also common. More proactive screening for TB comorbidity is required in obstetric and gynaecological wards.

Advances in multiparametric molecular diagnostics technologies for respiratory tract infections.

PURPOSE OF REVIEW: Respiratory tract infections (RTIs) are caused by a variety of bacterial, viral, fungal, and other pathogens and cause millions of deaths each year. Current standard microbiological culture-based tests are laborious and time consuming. Thus, patients are initially treated empirically, leading to inappropriate use of antibiotics. The purpose of this article is to provide clinicians and scientists with a review of recently available commercial multiparametric molecular diagnostics tests for the detection of RTIs so that they can be considered for use instead of, or in combination with, traditional culture techniques. RECENT FINDINGS: Several technologies have become commercially available for the multiparametric molecular detection of RTIs in the past decade including tests based on PCR-array, PCR-mass spectrometry, and multiplex qPCR technologies. The majority of these tests are for the detection of viruses, but more recently companies have begun to focus on detection of viruses, bacteria, and associated drug resistances in a single product to maximize the information provided to the clinician by a single test. SUMMARY: We describe the recent advances in commercial multiparametric molecular diagnostics technologies for the diagnosis of RTIs. Combining the specific and sensitive molecular detection of bacteria, viruses, fungi, and drug resistances is key if molecular methods are to replace traditional culture. The reliability of the molecular drug-resistance markers chosen, the need for the quantitative detection of some organisms, and throughput are also important considerations for new technology developers.

Trends in childhood tuberculosis in Zambia: a situation analysis.

OBJECTIVES: To ascertain childhood tuberculosis (TB) trends, human immunodeficiency virus (HIV) co-infection rates and multi-drug resistant TB (MDR-TB) prevalence rates in Zambia. METHODS: A retrospective review of Zambian annual TB notification data and National TB Programme reports for a 7 year period (2004-2011). TB trends were stratified by age and HIV status. RESULTS: The total number of children notified during this period with all forms of TB was 40 976. A total of 2670 of 40 976 (6%) were smear-positive cases. Notification rates of all forms of childhood TB show a decline in trends from 135 per 100 000 population in 2004, to 69 per 100 000 population in 2011. CONCLUSIONS: Childhood TB is an important but neglected problem in Zambia highlighted by the fact that no data exists on HIV co-infection and MDR-TB. Strengthening of the National TB Programme and diagnostics services/algorithms are required to accurately define the TB burden, HIV co-infection and MDR-TB rates in children in Zambia.

Tuberculosis and tuberculosis/HIV/AIDS-associated mortality in Africa: the urgent need to expand and invest in routine and research autopsies.

Frequently quoted statistics that tuberculosis and human immunodeficiency virus (HIV)/AIDS are the most important infectious causes of death in high-burden countries are based on clinical records, death certificates, and verbal autopsy studies. Causes of death ascertained through these methods are known to be grossly inaccurate. Most data from Africa on mortality and causes of death currently used by international agencies have come from verbal autopsy studies, which only provide inaccurate estimates of causes of death. Autopsy rates in most sub-Saharan African countries have declined over the years, and actual causes of deaths in the community and in hospitals in most sub-Saharan African countries remain unknown. The quality of cause-specific mortality statistics remains poor. The effect of various interventions to reduce mortality rates can only be evaluated accurately if cause-specific mortality data are available. Autopsy studies could have particular relevance to direct public health interventions, such as vaccination programs or preventive therapy, and could also allow for study of background levels of subclinical tuberculosis disease, Mycobacterium tuberculosis-HIV coinfection, and other infectious and noncommunicable diseases not yet clinically manifest. Autopsies performed soon after death may represent a unique opportunity to understand the pathogenesis of M. tuberculosis and the pathogenesis of early deaths after initiation of antiretroviral therapy. The few autopsies performed so far for research purposes have yielded invaluable information and insights into tuberculosis, HIV/AIDS, and other opportunistic infections. Accurate cause-specific mortality data are essential for prioritization of governmental and donor investments into health services to reduce morbidity and mortality from deadly infectious diseases such as tuberculosis and HIV/AIDS. There is an urgent need for reviving routine and research autopsies in sub-Saharan African countries.

Drug-resistant tuberculosis--current dilemmas, unanswered questions, challenges, and priority needs.

Tuberculosis was declared a global emergency by the World Health Organization (WHO) in 1993. Following the declaration and the promotion in 1995 of directly observed treatment short course (DOTS), a cost-effective strategy to contain the tuberculosis epidemic, nearly 7 million lives have been saved compared with the pre-DOTS era, high cure rates have been achieved in most countries worldwide, and the global incidence of tuberculosis has been in a slow decline since the early 2000s. However, the emergence and spread of multidrug-resistant (MDR) tuberculosis, extensively drug-resistant (XDR) tuberculosis, and more recently, totally drug-resistant tuberculosis pose a threat to global tuberculosis control. Multidrug-resistant tuberculosis is a man-made problem. Laboratory facilities for drug susceptibility testing are inadequate in most tuberculosis-endemic countries, especially in Africa; thus diagnosis is missed, routine surveillance is not implemented, and the actual numbers of global drug-resistant tuberculosis cases have yet to be estimated. This exposes an ominous situation and reveals an urgent need for commitment by national programs to health system improvement because the response to MDR tuberculosis requires strong health services in general. Multidrug-resistant tuberculosis and XDR tuberculosis greatly complicate patient management within resource-poor national tuberculosis programs, reducing treatment efficacy and increasing the cost of treatment to the extent that it could bankrupt healthcare financing in tuberculosis-endemic areas. Why, despite nearly 20 years of WHO-promoted activity and >12 years of MDR tuberculosis-specific activity, has the country response to the drug-resistant tuberculosis epidemic been so ineffectual? The current dilemmas, unanswered questions, operational issues, challenges, and priority needs for global drug resistance screening and surveillance, improved treatment regimens, and management of outcomes and prevention of DR tuberculosis are discussed.

Tuberculosis diagnostics and biomarkers: needs, challenges, recent advances, and opportunities.

Tuberculosis is unique among the major infectious diseases in that it lacks accurate rapid point-of-care diagnostic tests. Failure to control the spread of tuberculosis is largely due to our inability to detect and treat all infectious cases of pulmonary tuberculosis in a timely fashion, allowing continued Mycobacterium tuberculosis transmission within communities. Currently recommended gold-standard diagnostic tests for tuberculosis are laboratory based, and multiple investigations may be necessary over a period of weeks or months before a diagnosis is made. Several new diagnostic tests have recently become available for detecting active tuberculosis disease, screening for latent M. tuberculosis infection, and identifying drug-resistant strains of M. tuberculosis. However, progress toward a robust point-of-care test has been limited, and novel biomarker discovery remains challenging. In the absence of effective prevention strategies, high rates of early case detection and subsequent cure are required for global tuberculosis control. Early case detection is dependent on test accuracy, accessibility, cost, and complexity, but also depends on the political will and funder investment to deliver optimal, sustainable care to those worst affected by the tuberculosis and human immunodeficiency virus epidemics. This review highlights unanswered questions, challenges, recent advances, unresolved operational and technical issues, needs, and opportunities related to tuberculosis diagnostics.

The use of representative community samples to assess SARS-CoV-2 lineage competition: Alpha outcompetes Beta and wild-type in England from January to March 2021.

Genomic surveillance for SARS-CoV-2 lineages informs our understanding of possible future changes in transmissibility and vaccine efficacy and will be a high priority for public health for the foreseeable future. However, small changes in the frequency of one lineage over another are often difficult to interpret because surveillance samples are obtained using a variety of methods all of which are known to contain biases. As a case study, using an approach which is largely free of biases, we here describe lineage dynamics and phylogenetic relationships of the Alpha and Beta variant in England during the first 3 months of 2021 using sequences obtained from a random community sample who provided a throat and nose swab for rt-PCR as part of the REal-time Assessment of Community Transmission-1 (REACT-1) study. Overall, diversity decreased during the first quarter of 2021, with the Alpha variant (first identified in Kent) becoming predominant, driven by a reproduction number 0.3 higher than for the prior wild-type. During January, positive samples were more likely to be Alpha in those aged 18 to 54 years old. Although individuals infected with the Alpha variant were no more likely to report one or more classic COVID-19 symptoms compared to those infected with wild-type, they were more likely to be antibody-positive 6 weeks after infection. Further, viral load was higher in those infected with the Alpha variant as measured by cycle threshold (Ct) values. The presence of infections with non-imported Beta variant (first identified in South Africa) during January, but not during February or March, suggests initial establishment in the community followed by fade-out. However, this occurred during a period of stringent social distancing. These results highlight how sequence data from representative community surveys such as REACT-1 can augment routine genomic surveillance during periods of lineage diversity.

Evaluation of the Xpert MTB/RIF assay at a tertiary care referral hospital in a setting where tuberculosis and HIV infection are highly endemic.

BACKGROUND. There were 1.45 million deaths from tuberculosis in 2011. A substantial proportion of active pulmonary tuberculosis cases in countries where tuberculosis, human immunodeficiency virus (HIV) infection, and AIDS are highly endemic remain undiagnosed because of the reliance on sputum-smear microscopy. This study evaluated the performance of the Xpert MTB/RIF assay at a tertiary care referral center in Zambia, a country where the burden of tuberculosis and HIV infection is high. METHODS. A total of 881 adult inpatients admitted to University Teaching Hospital in Lusaka who were able to produce sputum were enrolled and analyzed in the study, irrespective of admission diagnosis. Sputum specimens were analyzed by fluorescence smear microscopy, the Xpert MTB/RIF assay, mycobacterial growth indicator tube (MGIT) culture,and MGIT drug-susceptibility testing. The sensitivity and specificity of the Xpert MTB/RIF assay were evaluated using culture as the gold standard. RESULTS. Culture-confirmed tuberculosis was found in 201 of 881 patients (22.8%). The specificity of the Xpert MTB/RIF assay was 95.0% (95% confidence interval [CI], 92.4%­96.8%),and the sensitivity was 86.1% (95% CI, 80.3%­90.4%). In sputum smear­negative, culture-positive cases, the assay was 74.7% sensitive (95% CI, 64.6%­82.8%), identifying 71 additional tuberculosis cases that were not detected by smear microscopy.A total of 18 of 111 patients with tuberculosis who were tested (16.2%) had multidrug-resistant (MDR) tuberculosis.The sensitivity and specificity of the Xpert MTB/RIF assay for detecting culture-confirmed, rifampicin-resistant tuberculosis was 81.3% (95% CI, 53.7%­95.0%) and 97.5% (95% CI,90.4%­99.6%), respectively. CONCLUSIONS. The Xpert MTB/RIF assay performs better than smear microscopy in an inpatient setting in a country where tuberculosis and HIV infection are highly endemic. Assessment of its usefulness and cost-effectiveness for increased detection of tuberculosis cases missed by sputum smear and for concomitant screening for MDR tuberculosis among adult inpatients attending tertiary care referral centers in other countries with a high burden of tuberculosis and HIV infection is warranted [corrected].

SeekTB, a two-stage multiplex real-time-PCR-based method for differentiation of the Mycobacterium tuberculosis complex.

Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). The accurate identification of the MTC member causing human infection is important because the treatment of TB caused by some MTC members requires an alteration of the standard drug regimen, it can inform whether transmission is human to human or zoonotic, and it enables accurate epidemiology studies that help improve TB control. In this study, an internally controlled two-stage multiplex real-time PCR-based method, SeekTB, was developed for the accurate identification of all members of the MTC. The method was tested against a panel of well-characterized bacterial strains (n = 180) and determined to be 100% specific for members of the MTC. Additionally, 125 Mycobacteria Growth Indicator Tube (MGIT)-positive cultures were blindly tested by using SeekTB, and the results were compared to those of the GenoType MTBC and TBc ID tests. The SeekTB and GenoType MTBC results were 100% concordant, identifying 84 of these isolates as M. tuberculosis isolates and 41 as non-MTC isolates. Nine discordant results between the molecular methods and the TBc ID culture confirmation test were observed; however, nucleotide sequencing confirmed the results obtained with GenoType MTBC and SeekTB. SeekTB is the first-described internally controlled multiplex real-time PCR diagnostic method for the accurate identification of all eight members of the MTC. This method, designed for use on cultured patient samples, is specific, sensitive, and rapid, with a turnaround time to results of approximately 1.5 to 3.5 h, depending on which, if any, member of the MTC is present.

Leprosy trends in Zambia 1991-2009.

OBJECTIVE: To document leprosy trends in Zambia over the past two decades to ascertain the importance of leprosy as a health problem in Zambia. METHODS: Retrospective study covering the period 1991-2009 of routine national leprosy surveillance data, published national programme review reports and desk reviews of in-country TB reports. RESULTS: Data reports were available for all the years under study apart from years 2001, 2002 and 2006. The Leprosy case notification rates (CNR) declined from 2.73/10 000 population in 1991 to 0.43/10 000 population in 2009. The general leprosy burden showed a downward trend for both adults and children. Leprosy case burden dropped from approximately 18 000 cases in 1980 to only about 1000 cases in 1996, and by the year 2000, the prevalence rates had fallen to 0.67/10 000 population. There were more multibacillary cases of leprosy than pauci-bacillary cases. Several major gaps in data recording, entry and surveillance were identified. Data on disaggregation by gender, HIV status or geographical origin were not available. CONCLUSION: Whilst Zambia has achieved WHO targets for leprosy control, leprosy prevalence data from Zambia may not reflect real situation because of poor data recording and surveillance. Greater investment into infrastructure and training are required for more accurate surveillance of leprosy in Zambia.