RATIONALE:Mycobacterium avium complex lung disease (MAC-LD) is clinically heterogeneous and carries diverse outcomes. OBJECTIVES:To describe predictors of clinical progression of MAC-LD in a state-wide cohort. METHODS:We enrolled adults with MAC-LD from across Virginia, USA starting in 2021. Every 6 months we performed respiratory quality of life questionnaire, scored CT scans, and recorded respiratory mycobacterial cultures including MAC speciation. Outcomes were classified using NTM-NET consensus definitions, factors predicting clinical progression analyzed by Poisson regression, and hierarchical clustering on principal components derived from Factorial Analysis of Mixed Data. MEASUREMENTS AND MAIN RESULTS:Of 105 participants the median follow-up was 917 days. Mean age was 69.8 years, 79 (75%) were women, and 70 (67%) had nodular bronchiectasis. M. intracellulare was the most common species, present in 48 (46%) participants at enrollment, followed by M. avium (29, 28%), and M. intracellulare subspecies chimaera (11, 10%). Only 2 (9%) of 22 evaluable participants met the NTM-NET definition of cure. In all participants after multivariable adjustment, older baseline age (incidence rate ratio 1.03 [1, 1.06], p = 0.04) and fibrocavitary CT scan pattern (2.57 [1.33, 4.96], p = 0.005), were associated with unfavorable 12-month clinical progression. Species type and species persistence contributed to characteristics of three distinct phenotypes of MAC-LD of varying severity and clinical progression. CONCLUSIONS:The majority of participants were not assessable for MAC-LD treatment outcomes using strict NTM-NET definitions. Species informed phenotypes of MAC lung disease are prognostically useful and can inform routine management and trial design.

Introduction Biannual (ie, every 6 months) mass drug administration of azithromycin has reduced childhood mortality in Niger, but its effects on specific respiratory pathogens are not fully elucidated.Methods Across 2 years of the Macrolides Oraux pour Réduire les Décès avec un Oeil sur la Résistance (MORDOR) study in Niger, we evaluated 1468 nasopharyngeal swabs from children who presented for care with respiratory symptoms. Swabs were tested by quantitative PCR using a customised TaqMan Array Card that included assays for 19 respiratory pathogens.Results Nasopharyngeal detection of Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae was common in both azithromycin and placebo communities. The prevalence was reduced in children from azithromycin communities for just two pathogens: Bordetella pertussis and H. influenzae type b (Hib). These children had a 49% and 65% reduction in the prevalence of B. pertussis and Hib in nasopharyngeal swabs, respectively, compared with children from the control communities (prevalence ratios 0.51, 95% CI 0.35, 0.75; and 0.35, 95% CI 0.17, 0.71).Conclusions Biannual administration of azithromycin to communities in Niger was associated with lower prevalence of B. pertussis and Hib compared with placebo. These reductions may explain some of the childhood mortality benefit of azithromycin.Trial registration number NCT02048007.

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers’ diarrhea. The vaccine candidate ETVAX encompasses several ETEC colonization factors (CFs) with a hybrid LT (heat-labile toxin)/cholera toxin B subunit adjuvanted with a double-mutant LT. Stool samples from a Phase 2b ETVAX trial were tested by a PCR-based customized TaqMan Array Card (TAC), including three ETEC toxin genes (LT and heat-stable toxins, STh and STp) and 18 ETEC CFs. Stool samples were also tested with the molecular platform Amplidiag and culture, followed by GM1-enzyme-linked immunosorbent assay (ELISA) and inhibition GM1-ELISA for LT and ST and dot blot for CFs of ETECs identified among six culture isolates (maximum). Compared with Amplidiag, TAC yielded 89.4% sensitivity (320/358) and 96.4% specificity (405/420) for ETEC detection. The two methods demonstrated a good quantitative correlation (quantification cycle R2 = 0.827, P < 0.05). Compared with culture, TAC and Amplidiag each exhibited 96.8% (184/190) sensitivity and identified an additional of 151 and 174 PCR positives in 588 culture-negative stools, respectively. The concordance of stool TAC versus ELISA of ETEC colonies for LT and STh/STp was 85.5% (165/193). TAC demonstrated 98% sensitivity and 92% specificity versus the dot blot results of 793 colonies for the ETVAX CFs CFA/I, CS3, CS5, and CS6. Overall ETEC was detected by TAC in 335 (43.1%) and by Amplidiag in 358 (46.0%) of specimens compared to 190 (24.4%) by culture. We conclude that molecular diagnostic approaches of TAC or Amplidiag increase the detection of ETEC compared with culture, and TAC can also provide vaccine-subtype ETEC data.CLINICAL TRIALSThis study was registered with ClinicalTrials.gov as NCT03729219.IMPORTANCEEnterotoxigenic Escherichia coli (ETEC) is an important cause of childhood and travelers’ diarrhea. Vaccines in development utilize specific toxins and colonization factors (CFs) as antigens. Therefore, clinical microbiologic diagnostic methods are needed to discriminate specific toxins and CFs, both for vaccine trials and to guide epidemiology. In this work, we assessed the diagnostic performance of several methods for ETEC: a PCR-based customized TaqMan Array Card (TAC) and the molecular platform Amplidiag on stool and E. coli culture, followed by GM1-enzyme-linked immunosorbent assay for toxins and dot blot for CFs. Stool samples from a Phase 2b ETEC vaccine trial were used. Overall, ETEC was detected by TAC in 335 (43.1%) and by Amplidiag in 358 samples (46.0%) compared to 190 (24.4%) by culture. TAC additionally provided CF data with 98% sensitivity and 92% specificity. We conclude that the molecular diagnostic approaches of TAC or Amplidiag increase the detection of ETEC compared with culture.

Background Quantitative polymerase chain reaction (qPCR) targeting ipaH has been proven to be highly efficient in detecting Shigella in clinical samples compared to culture-based methods, which underestimate Shigella burden by 2- to 3-fold. qPCR assays have also been developed for Shigella speciation and serotyping, which is critical for both vaccine development and evaluation.Methods The Enterics for Global Health (EFGH) Shigella surveillance study will utilize a customized real-time PCR-based TaqMan Array Card (TAC) interrogating 82 targets, for the detection and differentiation of Shigella spp, Shigella sonnei, Shigella flexneri serotypes, other diarrhea-associated enteropathogens, and antimicrobial resistance (AMR) genes. Total nucleic acid will be extracted from rectal swabs or stool samples, and assayed on TAC. Quantitative analysis will be performed to determine the likely attribution of Shigella and other particular etiologies of diarrhea using the quantification cycle cutoffs derived from previous studies. The qPCR results will be compared to conventional culture, serotyping, and phenotypic susceptibility approaches in EFGH.Conclusions TAC enables simultaneous detection of diarrheal etiologies, the principal pathogen subtypes, and AMR genes. The high sensitivity of the assay enables more accurate estimation of Shigella-attributed disease burden, which is critical to informing policy and in the design of future clinical trials. Shigella spp, Shigella flexneri serotypes, and other diarrhea-associated enteropathogens are detected and quantified with a customized real-time polymerase chain reaction-based TaqMan Array Card in the Enterics for Global Health: Shigella surveillance study. Shigella burden will be estimated with a quantitative methodology.

In the MORDOR I trial, children under 5 years of age were randomized to receive biannual (every 6 months) azithromycin for 2 years in Niger, Malawi, and Tanzania. In 30 Nigerien communities, children aged 7-11 years, who were not enrolled in the MORDOR I trial to receive biannual azithromycin, were assessed for carriage of seven respiratory pathogens. We aimed to see whether there were effects on the carriage of these seven respiratory pathogens among 3,187 children aged 7-11 years living in the 30 communities via nasopharyngeal swabs collected at baseline (N = 1,066), as well as at year 1 (N = 1,019) and year 2 (N = 1,102)-each about 6 months after azithromycin or placebo treatment of children under age five. Most children were positive for Haemophilus influenzae (baseline: 83.8%; interquartile range [IQR]: 78.7-90.4) and Streptococcus pneumoniae (baseline: 82.9%; IQR: 74.2-86.8) at all time points regardless of treatment group. There were no differences in prevalence nor quantity of H. influenzae (prevalence ratio: 0.95; 95% CI: 0.90, 1.02), S. pneumoniae (prevalence ratio: 1.01; 95% CI: 0.96, 1.07), or any of the other respiratory pathogens in the treatment versus control groups at any time point. S. pneumoniae serotype 6AB (7.7%) and Neisseria meningitidis serotype W135 (24.9%) were the most prevalent serotypes detected among all positive S. pneumoniae and N. meningitidis samples, respectively. Biannual azithromycin did not reduce carriage of respiratory pathogens 6 months after the most recent round of biannual azithromycin among older nontreated children (aged 7-11 years) living in treatment communities.

Background Virginia is a large state in the USA, yet it remains unclear what percentage of the population has had natural COVID-19 infection and whether risk factors for infection have changed over time. Methods Using a longitudinal cohort, from December 2021-July 2022 we performed follow up serology and a questionnaire on 784 individuals from across Virginia who had previously participated in a statewide COVID-19 seroepidemiology study in 2020. Children were also invited to participate and an additional 62 children also completed the study. Serology was performed using Roche nucleocapsid and spike serological assays. Results The majority of participants were white (78.6%), over 50 years old (60.9%), and reported having received COVID-19 vaccine (93.4%). 28.6% had evidence of prior COVID-19 infection (nucleocapsid positive). Reweighted by region, age, and sex to match the Virginia census data, the seroprevalence of nucleocapsid antibodies was estimated to be 30.6% (95% CI: 24.7, 36.6). We estimated that 25–53% of COVID-19 infections were asymptomatic. Infection rates were lower in individuals > 60 years old and were higher in Blacks and Hispanics. Infection rates were also higher in those without health insurance, in those with greater numbers of household children, and in those that reported a close contact or having undergone quarantine for COVID-19. Participants from Southwest Virginia had lower seropositivity (16.2%, 95% CI 6.5, 26.0) than other geographic regions. Boosted vaccinees had lower infection rates than non-boosted vaccinees. Frequenting indoor bars was a risk factor for infection, while frequently wearing an N95 mask was protective, though the estimates of association were imprecise. Infection rates were higher in children than adults (56.5% vs. 28.6%). Infection in the parent was a risk factor for child infection. Spike antibody levels declined with time since last vaccination, particularly in those that were vaccinated but not previously infected. Neutralizing antibody positivity was high (97–99%) for wild type, alpha, beta, gamma, delta, and omicron variants. Neutralizing antibody levels were higher in the follow-up survey compared to the first survey in 2020 and among individuals with evidence of natural infection compared to those without. Conclusions In this longitudinal statewide cohort we observed a lower-than-expected COVID-19 infection rate as of August 2022. Boosted vaccinees had lower infection rates. Children had higher infection rates and infections tracked within households. Previously identified demographic risk factors for infection tended to persist. Even after the omicron peak, a large number of Virginians remain uninfected with COVID-19, underscoring the need for ongoing vaccination strategies.

Quantitative polymerase chain reaction (qPCR) of dried blood spots (DBS) for pathogen detection is a potentially convenient method for infectious disease diagnosis. This study tested 115 DBS samples paired with whole blood specimens of children and adolescent from Burkina Faso, Sudan, and Madagascar by qPCR for a wide range of pathogens, including protozoans, helminths, fungi, bacteria, and viruses. Plasmodium spp. was consistently detected from DBS but yielded a mean cycle threshold (Ct) 5.7 ± 1.6 higher than that from whole blood samples. A DBS qPCR Ct cutoff of 27 yielded 94.1% sensitivity and 95.1% specificity against the whole blood qPCR cutoff of 21 that has been previously suggested for malaria diagnosis. For other pathogens investigated, DBS testing yielded a sensitivity of only 8.5% but a specificity of 98.6% compared with whole blood qPCR. In sum, direct PCR of DBS had reasonable performance for Plasmodium but requires further investigation for the other pathogens assessed in this study.

BACKGROUND:Early detection and correction of low fluoroquinolone exposure may improve treatment of MDR-TB. OBJECTIVES:To explore a recently developed portable, battery-powered, UV spectrophotometer for measuring levofloxacin in saliva of people treated for MDR-TB. METHODS:Patients treated with levofloxacin as part of a regimen for MDR-TB in Northern Tanzania had serum and saliva collected concurrently at 1 and 4 h after 2 weeks of observed levofloxacin administration. Saliva levofloxacin concentrations were quantified in the field via spectrophotometry, while serum was analysed at a regional laboratory using HPLC. A Bayesian population pharmacokinetics model was used to estimate the area under the concentration-time curve (AUC0-24). Subtarget exposures of levofloxacin were defined by serum AUC0-24 <80 mg·h/L. The study was registered at Clinicaltrials.gov with clinical trial identifier NCT04124055. RESULTS:Among 45 patients, 11 (25.6%) were women and 16 (37.2%) were living with HIV. Median AUC0-24 in serum was 140 (IQR = 102.4-179.09) mg·h/L and median AUC0-24 in saliva was 97.10 (IQR = 74.80-121.10) mg·h/L. A positive linear correlation was observed with serum and saliva AUC0-24, and a receiver operating characteristic curve constructed to detect serum AUC0-24 below 80 mg·h/L demonstrated excellent prediction [AUC 0.80 (95% CI = 0.62-0.94)]. Utilizing a saliva AUC0-24 cut-off of 91.6 mg·h/L, the assay was 88.9% sensitive and 69.4% specific in detecting subtarget serum AUC0-24 values, including identifying eight of nine patients below target. CONCLUSIONS:Portable UV spectrophotometry as a point-of-care screen for subtarget levofloxacin exposure was feasible. Use for triage to other investigation or personalized dosing strategy should be tested in a randomized study.

Background:Levofloxacin is a preferred drug for multidrug-resistant (MDR)-tuberculosis (TB) with bactericidal activity that correlates with the pharmacokinetic exposures of serum peak concentration (Cmax) and total area under the concentration time curve (AUC0-24). Pharmacokinetic exposures can be measured to personalize dosing to reach targets, but this practice requires venepuncture, chromatographic or mass spectrometry equipment, and technical expertise. We sought to demonstrate the accuracy of using urine colorimetry as a more feasible estimation of levofloxacin exposure. Method:A colorimetric method using bromocresol green was tested on spiked urine samples with levofloxacin measured using a spectrophotometer. This method was tested in urine samples of healthy volunteers given one 750 mg dose of levofloxacin with urine collected at 0-4 h, 4-8 h, and 8-24 h intervals, and concomitant serum samples were collected and analyzed by high-performance liquid chromatography. Validation of this assay was done in a cohort of people living with human immunodeficiency virus (PLWH), initiating a levofloxacin containing MDR-TB regimen. Results:Urine colorimetry was reproducible in spiked samples and the calibration was curve linear for levofloxacin concentrations ranging from 7.8 μg/ml to 250 μg/ml, with r = 0.98. In healthy volunteers, correlation between urine absorbance values and serum AUC0-24 was highest in urine collected between 4 and 8 h (r = 0.91, P = 0.01), yet in PLWH, urine collected between 0 and 4 h had highest correlation (r = 0.66, P = 0.05). The area under the receiver operating characteristics curve was >0.8 in the derivation, as well as the validation cohort for the urine absorbance values identifying people with total serum exposure below target. Conclusion:Urine colorimetry was highly sensitive in predicting target serum concentrations. Colorimetric methods to determine levofloxacin in urine may improve the feasibility of therapeutic drug monitoring and personalized dose adjustment in TB endemic settings.

Background Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials. Methods We compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, qPCR-attributable Shigella (defined by an ipaH gene cycle threshold <27.9). Among Shigella MSD cases, we determined risk factors for death and derived a clinical severity score. Results Compared to culture-positive Shigella MSD cases (n=745), culture-negative/qPCR-attributable Shigella cases (n=852) were more likely to be under 12 months, stunted, have a longer duration of diarrhea, and less likely to have high stool frequency or a fever. There …

Background. Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials. Methods. We compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, quantitative polymerase chain reaction (qPCR)-attributable Shigella (defined by an ipaH gene cycle threshold <27.9). Among Shigella MSD cases, we determined risk factors for death and derived a clinical severity score. Results. Compared to culture- positive Shigella MSD cases (n = 745), culture- negative/qPCR-attributable Shigella cases (n = 852) were more likely to be under 12 months, stunted, have a longer duration of diarrhea, and less likely to have high stool frequency or a fever. There was no difference in dehydration, hospitalization, or severe classification from a modified Vesikari score. Twenty-two ( 1.8%) Shigella MSD cases died within the 14-days after presentation to health facilities, and 59.1% of these deaths were in culture-negative cases. Age <12 months, diarrhea duration prior to presentation, vomiting, stunting, wasting, and hospitalization were associated with mortality. A model-derived score assigned points for dehydration, hospital admission, and longer diarrhea duration but was not significantly better at predicting 14- day mortality than a modified Vesikari score. Conclusions. A composite severity score consistent with severe disease or dysentery may be a pragmatic clinical endpoint for severe shigellosis in vaccine trials. Reliance on culture for microbiologic confirmation may miss a substantial number of Shigella cases but is currently required to measure serotype specific immunity.

Antimicrobial resistance (AMR) is an emerging public health problem and methods for surveillance are needed. We designed 85 sequence-specific PCR reactions to detect 79 genes or mutations associated with resistance across 10 major antimicrobial classes, with a focus on E. coli. The 85 qPCR assays demonstrated >99.9% concordance with sequencing. We evaluated the correlation between genotypic resistance markers and phenotypic susceptibility results on 239 E. coli isolates. Both sensitivity and specificity exceeded 90% for ampicillin, ceftriaxone, cefepime, imipenem, ciprofloxacin, azithromycin, gentamicin, amikacin, trimethoprim/sulfamethoxazole, tetracycline, and chloramphenicol phenotypic susceptibility results. We then evaluated the assays on direct stool specimens and observed a sensitivity of 97% ± 5 but, as expected, a lower specificity of 75% ± 31 versus the genotype of the E. coli cultured from stool. Finally, the assays were incorporated into a convenient TaqMan Array Card (TAC) format. These assays may be useful for tracking AMR in E. coli isolates or directly in stool for targeted testing of the fecal antibiotic resistome.

BackgroundOptimum management of childhood diarrhoea in low-resource settings has been hampered by insufficient data on aetiology, burden, and associated clinical characteristics. We used quantitative diagnostic methods to reassess and refine estimates of diarrhoea aetiology from the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study.MethodsWe re-analysed stool specimens from the multisite MAL-ED cohort study of children aged 0–2 years done at eight locations (Dhaka, Bangladesh; Vellore, India; Bhaktapur, Nepal; Naushero Feroze, Pakistan; Venda, South Africa; Haydom, Tanzania; Fortaleza, Brazil; and Loreto, Peru), which included active surveillance for diarrhoea and routine non-diarrhoeal stool collection. We used quantitative PCR to test for 29 enteropathogens, calculated population-level …

Background Enteropathogen infections in early childhood not only cause diarrhoea but contribute to poor growth. We used molecular diagnostics to assess whether particular enteropathogens were associated with linear growth across seven low-resource settings. Methods We used quantitative PCR to detect 29 enteropathogens in diarrhoeal and non-diarrhoeal stools collected from children in the first 2 years of life obtained during the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) multisite cohort study. Length was measured monthly. We estimated associations between aetiology-specific diarrhoea and subclinical enteropathogen infection and quantity and attained length in 3 month intervals, at age 2 and 5 years, and used a longitudinal model to account for temporality and time-dependent confounding. Findings Among 1469 children who completed 2 year follow-up, 35 622 stool samples were tested and yielded valid results. Diarrhoea! episodes attributed to bacteria and parasites, but not viruses, were associated with small decreases in length after 3 months and at age 2 years. Substantial decrements in length at 2 years were associated with subdinical, non-diarrhoeal, infection with Shigella (length-for-age Z score [LAZ] reduction -0.14, 95% CI -0.27 to -0.01), enteroaggregative Escherichia coli -0.37 to -0.05), Campylobacter (-0.17, -0.32 to -0.01), and Giardia (-0.17, -0.30 to -0.05). Norovirus, Cryptosporidium, typical enteropathogenic E coil, and Enterocytozoon bieneusi were also associated with small decrements in LAZ. Shigeila and E bieneusi were associated with the largest decreases in LAZ per log increase in quantity per g of stool (-0.13 LAZ, 95% CI -0.22 to -0.03 for Shigella; -0.14, -0.26 to -0.02 for E bieneusi). Based on these models, interventions that successfully decrease exposure to Shigella, enteroaggregative E coli, Campylobacter, and Giardia could increase mean length of children by 0.12-0-37 LAZ (0.4-1-2 cm) at the MAL-ED sites. Interpretation Subclinical infection and quantity of pathogens, particularly Shigeila, enteroaggregative E coil, Campylobacter, and Giardia, had a substantial negative association with linear growth, which was sustained during the first 2 years of life, and in some cases, to 5 years. Successfully reducing exposure to certain pathogens might reduce global stunting. Copyright (C) 2018 The Author(s). Published by Elsevier Ltd.

Background Knowledge of causes of sepsis in sub-Saharan Africa is limited. A better understanding of the microbiology of bloodstream infections could improve outcomes. Methods We used a quantitative polymerase chain reaction (qPCR)-based TaqMan Array Card (TAC) to directly test for 43 targets from whole blood. We analyzed 336 cryopreserved specimens from adult Ugandans with sepsis enrolled in a multisite study; 84% were infected with human immunodeficiency virus. We compared qPCR TAC results with blood culture and determined the association of qPCR with study participant outcomes using logistic regression. Results The most frequently detected targets were cytomegalovirus (CMV, n = 139, 41%), Mycobacterium tuberculosis (TB, n = 70, 21%), Plasmodium (n = 35, 10%), and Streptococcus pneumoniae (n = 31, 9%). Diagnostic performance varied by target with qPCR sensitivity averaging 61 ± 28% and specificity 98 ± 3% versus culture. In multivariable analysis, independent factors associated with in-hospital mortality included CMV viremia (adjusted odds ratio [aOR] 3.2, 95% confidence interval [CI], 1.8-5.5; p < .01) and TB qPCR-positivity, whether blood culture-positive (aOR 4.6, 95% CI, 2.1-10.0; p < .01) or blood culture-negative (aOR 2.9, 95% CI, 1.2-6.9; p = .02). Conclusions Using qPCR TAC on direct blood specimens, CMV and TB were the most commonly identified targets and were independently associated with increased in-hospital mortality. qPCR TAC screening of blood for multiple targets may be useful to guide triage and treatment of sepsis in sub-Saharan Africa.

BACKGROUND:Optimum management of childhood diarrhoea in low-resource settings has been hampered by insufficient data on aetiology, burden, and associated clinical characteristics. We used quantitative diagnostic methods to reassess and refine estimates of diarrhoea aetiology from the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study. METHODS:We re-analysed stool specimens from the multisite MAL-ED cohort study of children aged 0-2 years done at eight locations (Dhaka, Bangladesh; Vellore, India; Bhaktapur, Nepal; Naushero Feroze, Pakistan; Venda, South Africa; Haydom, Tanzania; Fortaleza, Brazil; and Loreto, Peru), which included active surveillance for diarrhoea and routine non-diarrhoeal stool collection. We used quantitative PCR to test for 29 enteropathogens, calculated population-level pathogen-specific attributable burdens, derived stringent quantitative cutoffs to identify aetiology for individual episodes, and created aetiology prediction scores using clinical characteristics. FINDINGS:We analysed 6625 diarrhoeal and 30 968 non-diarrhoeal surveillance stools from 1715 children. Overall, 64·9% of diarrhoea episodes (95% CI 62·6-71·2) could be attributed to an aetiology by quantitative PCR compared with 32·8% (30·8-38·7) using the original study microbiology. Viral diarrhoea (36·4% of overall incidence, 95% CI 33·6-39·5) was more common than bacterial (25·0%, 23·4-28·4) and parasitic diarrhoea (3·5%, 3·0-5·2). Ten pathogens accounted for 95·7% of attributable diarrhoea: Shigella (26·1 attributable episodes per 100 child-years, 95% CI 23·8-29·9), sapovirus (22·8, 18·9-27·5), rotavirus (20·7, 18·8-23·0), adenovirus 40/41 (19·0, 16·8-23·0), enterotoxigenic Escherichia coli (18·8, 16·5-23·8), norovirus (15·4, 13·5-20·1), astrovirus (15·0, 12·0-19·5), Campylobacter jejuni or C coli (12·1, 8·5-17·2), Cryptosporidium (5·8, 4·3-8·3), and typical enteropathogenic E coli (5·4, 2·8-9·3). 86·2% of the attributable incidence for Shigella was non-dysenteric. A prediction score for shigellosis was more accurate (sensitivity 50·4% [95% CI 46·7-54·1], specificity 84·0% [83·0-84·9]) than current guidelines, which recommend treatment only of bloody diarrhoea to cover Shigella (sensitivity 14·5% [95% CI 12·1-17·3], specificity 96·5% [96·0-97·0]). INTERPRETATION:Quantitative molecular diagnostics improved estimates of pathogen-specific burdens of childhood diarrhoea in the community setting. Viral causes predominated, including a substantial burden of sapovirus; however, Shigella had the highest overall burden with a high incidence in the second year of life. These data could improve the management of diarrhoea in these low-resource settings. FUNDING:Bill & Melinda Gates Foundation.

Antimalarial drug resistance exacerbates the global disease burden and complicates eradication efforts. To facilitate the surveillance of resistance markers in countries of malaria endemicity, we developed a suite of TaqMan assays for known resistance markers and compartmentalized them into a single array card (TaqMan array card, TAC). We included 87 assays for species identification, for the detection of Plasmodium falciparum mutations associated with chloroquine, atovaquone, pyrimethamine, sulfadoxine, and artemisinin resistance, and for neutral single nucleotide polymorphism (SNP) genotyping. Assay performance was first optimized using DNA from common laboratory parasite lines and plasmid controls. The limit of detection was 0.1 to 10 pg of DNA and yielded 100% accuracy compared to sequencing. The tool was then evaluated on 87 clinical blood samples from around the world, and the malaria TAC once again achieved 100% accuracy compared to sequencing and in addition detected the presence of mixed infections in clinical samples. With its streamlined protocol and high accuracy, this malaria TAC should be a useful tool for large-scale antimalarial resistance surveillance.

Background Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS).Methods GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children.Findings We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni or C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1.5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89.3% (95% CI 83.2-96.0) at the population level, compared with 51.5% (48.0-55.0) in the original GEMS analysis. The top six pathogens accounted for 77.8% (74.6-80.9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42.5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38.9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections.Interpretation A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised.

ABSTRACT We studied the significance of particular eis mutations on Mycobacterium tuberculosis drug resistance using a specialized transduction strategy. Recombinant strains harboring eis promoter mutations C14T, C12T, and G10A exhibited kanamycin resistance with MICs of 40, 10, and 20 μg/ml, respectively, while recombinant strains harboring C14G and C15G mutations were kanamycin susceptible (MIC, 2.5 to 5 μg/ml). Each of the eis mutants tested remained amikacin susceptible (MIC, 0.5 to 4 μg/ml). The identification of specific eis mutations is needed for accurate genotypic susceptibility testing for kanamycin.