Risk factors and titers of COVID-19 infection in a longitudinal statewide seroepidemiology cohort.

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.

The Clinical Presentation of Culture-positive and Culture-negative, Quantitative Polymerase Chain Reaction (qPCR)-Attributable Shigellosis in the Global Enteric Multicenter Study and Derivation of a Shigella Severity Score: Implications for Pediatric Shigella Vaccine Trials.

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.

Levofloxacin pharmacokinetics in saliva as measured by a mobile microvolume UV spectrophotometer among people treated for rifampicin-resistant TB in Tanzania.

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.

Etiology of Sepsis in Uganda Using a Quantitative Polymerase Chain Reaction-based TaqMan Array Card.

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.

The Malaria TaqMan Array Card Includes 87 Assays for Plasmodium falciparum Drug Resistance, Identification of Species, and Genotyping in a Single Reaction.

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.

Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study.

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 o 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. FUNDING: Bill & Melinda Gates Foundation.

eis Promoter C14G and C15G Mutations Do Not Confer Kanamycin Resistance in Mycobacterium tuberculosis.

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.

Microbiologic methods utilized in the MAL-ED cohort study.

A central hypothesis of The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) study is that enteropathogens contribute to growth faltering. To examine this question, the MAL-ED network of investigators set out to achieve 3 goals: (1) develop harmonized protocols to test for a diverse range of enteropathogens, (2) provide quality-assured and comparable results from 8 global sites, and (3) achieve maximum laboratory throughput and minimum cost. This paper describes the rationale for the microbiologic assays chosen and methodologies used to accomplish the 3 goals.

Plasma drug activity in patients on treatment for multidrug-resistant tuberculosis.

Little is known about plasma drug concentrations relative to quantitative susceptibility in patients with multidrug-resistant tuberculosis (MDR-TB). We previously described a TB drug activity (TDA) assay that determines the ratio of the time to detection of plasma-cocultured Mycobacterium tuberculosis versus control growth in a Bactec MGIT system. Here, we assess the activity of individual drugs in a typical MDR-TB regimen using the TDA assay. We also examined the relationship of the TDA to the drug concentration at 2 h (C2) and the MICs among adults on a MDR-TB regimen in Tanzania. These parameters were also compared to the treatment outcome of sputum culture conversion. Individually, moxifloxacin yielded superior TDA results versus ofloxacin, and only moxifloxacin and amikacin yielded TDAs equivalent to a -2-log killing. In the 25 patients enrolled on a regimen of kanamycin, levofloxacin, ethionamide, pyrazinamide, and cycloserine, the C2 values were found to be below the expected range for levofloxacin in 13 (52%) and kanamycin in 10 (40%). Three subjects with the lowest TDA result (<1.5, a finding indicative of poor killing) had significantly lower kanamycin C2/MIC ratios than subjects with a TDA of ≥1.5 (9.8 ± 8.7 versus 27.0 ± 19.1; P = 0.04). The mean TDAs were 2.52 ± 0.76 in subjects converting to negative in ≤2 months and 1.88 ± 0.57 in subjects converting to negative in >2 months (P = 0.08). In Tanzania, MDR-TB drug concentrations were frequently low, and a wide concentration/MIC range was observed that affected plasma drug activity ex vivo. An opportunity exists for pharmacokinetic optimization in current MDR-TB regimens, which may improve treatment response.

Discordance across several methods for drug susceptibility testing of drug-resistant Mycobacterium tuberculosis isolates in a single laboratory.

Given the increases in drug-resistant tuberculosis, laboratory capacities for drug susceptibility testing are being scaled up worldwide. A laboratory must decide among several endorsed methodologies. We evaluated 87 Mycobacterium tuberculosis isolates for concordance of susceptibility results across six methods: the L-J proportion method, MGIT 960 SIRE AST, Gene/Xpert MTB/RIF, GenoType MTBDRplus line probe assay, MycoTB MIC plate, and a laboratory-developed mycobacteriophage quantitative PCR (qPCR)-based method. Most (80%) isolates were multidrug resistant. Of the culture-based methods, the mycobacteriophage qPCR method was fastest, the L-J proportion method was the slowest, and the MGIT method required the most repeat testing (P < 0.05). For isoniazid (INH), 82% of isolates were susceptible by all methods or resistant by all methods, whereas for rifampin (RIF), ethambutol (EMB), and streptomycin (STR), such complete concordance was observed in 77%, 50%, and 51% of isolates, respectively (P < 0.05 for INH or RIF versus EMB or STR). The discrepancies of EMB and STR stemmed largely from diminished concordance of the MGIT EMB results (kappa coefficient range, 0.26 to 0.30) and the L-J STR result (kappa range, 0.35 to 0.45) versus other methods. Phage qPCR and the MycoTB MIC plate were the only methods that yielded second-line susceptibilities and revealed significant quantitative correlations for all drugs except cycloserine, as well as moderate to excellent kappa coefficients for all drugs except for para-aminosalicylic acid. In summary, the performance of M. tuberculosis susceptibility testing differs by platform and by drug. Laboratories should carefully consider these factors before choosing one methodology, particularly in settings where EMB and STR results are clinically important.

Shigella Detection and Molecular Serotyping With a Customized TaqMan Array Card in the Enterics for Global Health (EFGH): Shigella Surveillance Study.

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.

Application of quantitative second-line drug susceptibility testing at a multidrug-resistant tuberculosis hospital in Tanzania.

BACKGROUND: Lack of rapid and reliable susceptibility testing for second-line drugs used in the treatment of multidrug-resistant tuberculosis (MDR-TB) may limit treatment success. METHODS: Mycobacterium tuberculosis isolates from patients referred to Kibong'oto National TB Hospital in Tanzania for second-line TB treatment underwent confirmatory speciation and susceptibility testing. Minimum inhibitory concentration (MIC) testing on MYCOTB Sensititre plates was performed for all drugs available in the second-line formulary. We chose to categorize isolates as borderline susceptible if the MIC was at or one dilution lower than the resistance breakpoint. M. tuberculosis DNA was sequenced for resistance mutations in rpoB (rifampin), inhA (isoniazid, ethionamide), katG (isoniazid), embB (ethambutol), gyrA (fluoroquinolones), rrs (amikacin, kanamycin, capreomycin), eis (kanamycin) and pncA (pyrazinamide). RESULTS: Of 22 isolates from patients referred for second-line TB treatment, 13 (59%) were MDR-TB and the remainder had other resistance patterns. MIC testing identified 3 (14%) isolates resistant to ethionamide and another 8 (36%) with borderline susceptibility. No isolate had ofloxacin resistance, but 10 (45%) were borderline susceptible. Amikacin was fully susceptible in 15 (68%) compared to only 11 (50%) for kanamycin. Resistance mutations were absent in gyrA, rrs or eis for all 13 isolates available for sequencing, but pncA mutation resultant in amino acid change or stop codon was present in 6 (46%). Ten (77%) of MDR-TB patients had at least one medication that could have logically been modified based on these results (median 2; maximum 4). The most common modifications were a change from ethioniamide to para-aminosalicylic acid, and the use of higher dose levofloxacin. CONCLUSIONS: In Tanzania, quantitative second-line susceptibility testing could inform and alter MDR-TB management independent of drug-resistance mutations. Further operational studies are warranted.

Effect of Biannual Azithromycin to Children under 5 Years on the Carriage of Respiratory Pathogens among Children Aged 7-11 Years.

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.

Dual probe DNA capture for sensitive real-time PCR detection of Cryptosporidium and Giardia.

Nucleic acid amplification for the enteropathogens Cryptosporidium and Giardia is complicated by low target template concentrations and PCR inhibitors. In this work we designed dual capture oligonucleotides for both Cryptosporidium and Giardia 18S rRNA targets which when utilized during DNA extraction from stool improved the limit of detection of our multiplex PCR assay by 1-2 logs, to as little as 10 cysts. When applied to clinical specimens, the method improved the real-time PCR C(T) by an average of 10.7 ± 9.7 cycles. This work provides a highly sensitive protocol for Cryptosporidium and Giardia when limit of detection is of utmost importance.

Rapid first- and second-line drug susceptibility assay for Mycobacterium tuberculosis isolates by use of quantitative PCR.

The slow turnaround time for Mycobacterium tuberculosis drug susceptibility results is a barrier to care. We developed a rapid quantitative PCR (qPCR)-based phenotypic antimicrobial susceptibility test that utilizes amplification of the M. tuberculosis 16S rRNA gene after 3 days of incubation with antituberculosis drugs. To decrease background from killed organisms, we used propidium monoazide (PMA), a DNA-binding dye that penetrates damaged bacterial cells and renders DNA unamplifiable. M. tuberculosis was cultured in broth media containing PMA with or without drugs for 3 days prior to DNA extraction and real-time PCR amplification. 16S rRNA qPCR exhibited a significant decrease in threshold cycle (C(T)) time values (C(T) control - C(T) drug treated) with drug-susceptible strains compared with resistant strains. Susceptibility data were reported as ΔCT or as 2(Δ)(CT) and with appropriate cutoffs yielded an accuracy of 89 to 100% on 38 susceptible, multidrug-resistant, and extensively drug-resistant strains compared with conventional agar proportion susceptibility results for isoniazid, rifampin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide, para-aminosalicylic acid, linezolid, and cycloserine and compared with Bactec MGIT results for pyrazinamide. This PMA-qPCR assay is useful as a rapid 3-day first- and second-line drug susceptibility test for M. tuberculosis.

The genome of the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

Detection of Pathogens of Acute Febrile Illness Using Polymerase Chain Reaction from Dried Blood Spots.

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.

Urine colorimetry for levofloxacin pharmacokinetics and personalized dosing in people with drug-resistant tuberculosis.

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.

Genotypic antimicrobial resistance assays for use on E. coli isolates and stool specimens.

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.

Use of quantitative molecular diagnostic methods to investigate the effect of enteropathogen infections on linear growth in children in low-resource settings: longitudinal analysis of results from the MAL-ED cohort study.

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. Diarrhoeal 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 subclinical, non-diarrhoeal, infection with Shigella (length-for-age Z score [LAZ] reduction -0·14, 95% CI -0·27 to -0·01), enteroaggregative Escherichia coli (-0·21, -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 coli, and Enterocytozoon bieneusi were also associated with small decrements in LAZ. Shigella 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 Shigella, enteroaggregative E coli, 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. FUNDING: Bill & Melinda Gates Foundation.