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1.  Discordance across Several Methods for Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates in a Single Laboratory 
Journal of Clinical Microbiology  2014;52(1):156-163.
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.
doi:10.1128/JCM.02378-13
PMCID: PMC3911413  PMID: 24172155
2.  A Laboratory-Developed TaqMan Array Card for Simultaneous Detection of 19 Enteropathogens 
Journal of Clinical Microbiology  2013;51(2):472-480.
The TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here we developed an enteric TaqMan Array Card to detect 19 enteropathogens, including viruses (adenovirus, astrovirus, norovirus GII, rotavirus, and sapovirus), bacteria (Campylobacter jejuni/C. coli, Clostridium difficile, Salmonella, Vibrio cholerae, diarrheagenic Escherichia coli strains including enteroaggregative E. coli [EAEC], enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], and Shiga-toxigenic E. coli [STEC]), Shigella/enteroinvasive E. coli (EIEC), protozoa (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica), and helminths (Ascaris lumbricoides and Trichuris trichiura), as well as two extrinsic controls to monitor extraction and amplification efficiency (the bacteriophage MS2 and phocine herpesvirus). Primers and probes were newly designed or adapted from published sources and spotted onto microfluidic cards. Fecal samples were spiked with extrinsic controls, and DNA and RNA were extracted using the QiaAmp Stool DNA minikit and the QuickGene RNA Tissue kit, respectively, and then mixed with Ag-Path-ID One Step real-time reverse transcription-PCR (RT-PCR) reagents and loaded into cards. PCR efficiencies were between 90% and 105%, with linearities of 0.988 to 1. The limit of detection of the assays in the TAC was within a 10-fold difference from the cognate assays performed on plates. Precision testing demonstrated a coefficient of variation of below 5% within a run and 14% between runs. Accuracy was evaluated for 109 selected clinical specimens and revealed an average sensitivity and specificity of 85% and 77%, respectively, compared with conventional methods (including microscopy, culture, and immunoassay) and 98% and 96%, respectively, compared with our laboratory-developed PCR-Luminex assays. This TAC allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes.
doi:10.1128/JCM.02658-12
PMCID: PMC3553916  PMID: 23175269
3.  Febrile illness and pro-inflammatory cytokines are associated with lower neurodevelopmental scores in Bangladeshi infants living in poverty 
BMC Pediatrics  2014;14:50.
Background
An estimated one-third of children younger than 5 years in low- and middle-income countries fail to meet their full developmental potential. The first year of life is a period of critical brain development and is also when most of the morbidity from infection is suffered. We aimed to determine if clinical and biological markers of inflammation in the first year of life predict cognitive, language, and motor outcomes in children living in an urban slum in Bangladesh.
Methods
Children living in Dhaka, Bangladesh were observed from birth until 24 months of age. Febrile illness was used as a clinical marker of inflammation and elevated concentrations of inflammation-related cytokines (IL-1β, IL-6, TNF-α, IL-4, IL-10) in sera collected from a subset of the cohort (N = 127) at 6 months of age were used as biomarkers of inflammation. Psychologists assessed cognitive, language, and motor development using a culturally adapted version of the Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III) at 12 (N = 398) and 24 months of age (N = 210). We tested for the ability of febrile illness and elevated cytokine levels to predict developmental outcomes, independent of known predictors of stunting, family income, and maternal education.
Results
Every additional 10 days of fever was associated with a 1.9 decrease in language composite score and a 2.1 decrease in motor composite score (p = 0.005 and 0.0002, respectively). Elevated levels of the pro-inflammatory cytokines IL-1β (> 7.06 pg/mL) and IL-6 (> 10.52 pg/mL) were significantly associated with a 4.9 and 4.3 decrease in motor score, respectively. Conversely, an elevated level of the Th-2 cytokine IL-4 (> 0.70 pg/mL) was associated with a 3.6 increase in cognitive score (all p < 0.05).
Conclusions
Clinical and biological markers of inflammation in the first year of life were significantly associated with poor neurodevelopmental outcomes. Conversely, a Th2-like response was associated with a better outcome. These findings suggest that markers of inflammation could serve as prognostic indicators and potentially lead to immune-based therapies to prevent developmental delays in at-risk children.
doi:10.1186/1471-2431-14-50
PMCID: PMC3936797  PMID: 24548288
IL-1β; IL-6; IL-4; Child development; Cognition; Fever; Inflammation; Motor; Neurodevelopment; Pro-inflammatory
4.  Quantitative PCR for Detection of Shigella Improves Ascertainment of Shigella Burden in Children with Moderate-to-Severe Diarrhea in Low-Income Countries 
Journal of Clinical Microbiology  2013;51(6):1740-1746.
Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 104 ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 104 ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥2.9 × 104 ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 104 ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 104 ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.
doi:10.1128/JCM.02713-12
PMCID: PMC3716050  PMID: 23536399
5.  Early Therapeutic Drug Monitoring for Isoniazid and Rifampin among Diabetics with Newly Diagnosed Tuberculosis in Virginia, USA 
Slow responders to tuberculosis (TB) treatment in Virginia have prolonged treatment duration and consume more programmatic resources. Diabetes is an independent risk factor for slow response and low serum anti-TB drug concentrations. Thus, a statewide initiative of early therapeutic drug monitoring (TDM) for isoniazid and rifampin at 2 weeks after TB treatment was piloted for all diabetics with newly diagnosed TB. During the period of early TDM, 12/01/2011–12/31/2012, 21 diabetics had C2 hr concentrations performed and 16 (76%) had a value below the expected range for isoniazid, rifampin, or both. Fifteen had follow-up concentrations after dose adjustment and 12 (80%) increased to within the expected range (including all for rifampin). Of 16 diabetic patients with pulmonary TB that had early TDM, 14 (88%) converted their sputum culture to negative in <2 months. Early TDM for diabetics was operationally feasible, may speed response to TB therapy, and can be considered for TB programs with high diabetes prevalence.
doi:10.1155/2013/129723
PMCID: PMC3855970  PMID: 24349775
6.  Primary Multidrug-Resistant Mycobacterium tuberculosis in 2 Regions, Eastern Siberia, Russian Federation 
Emerging Infectious Diseases  2013;19(10):1649-1652.
Of 235 Mycobacterium tuberculosis isolates from patients who had not received tuberculosis treatment in the Irkutsk oblast and the Sakha Republic (Yakutia), eastern Siberia, 61 (26%) were multidrug resistant. A novel strain, S 256, clustered among these isolates and carried eis-related kanamycin resistance, indicating a need for locally informed diagnosis and treatment strategies.
doi:10.3201/eid1910.121108
PMCID: PMC3810730  PMID: 24047678
tuberculosis; multidrug-resistant tuberculosis; MDR TB; HIV; Siberia; Russian Federation; genotype; mycobacterial interspersed repetitive unit; MIRU-VNTR; pncA; pyrazinamide; tuberculosis and other mycobacteria
7.  Giardia intestinalis Assemblages A and B Infections in Nepal 
Giardia intestinalis is comprised of two major genotypes, A and B, which may vary in their propensity to cause disease. We tested for the presence of these two genotypes in stool samples from patients with gastrointestinal symptoms in Nepal. A total of 1,096 clinical specimens were screened by microscopy, and 45 samples with G. intestinalis were identified. Giardia infection was confirmed in 35 of 45 samples by a Giardia specific real-time polymerase chain reaction (PCR) assay. Genotyping of the Giardia PCR product by restriction fragment length polymorphism indicated that 74% (26 of 35) were assemblage B, 20% (7 of 35) were assemblage A, and 6% (2 of 35) were mixed assemblages.
PMCID: PMC3412867  PMID: 19706929
8.  Application of quantitative second-line drug susceptibility testing at a multidrug-resistant tuberculosis hospital in Tanzania 
BMC Infectious Diseases  2013;13:432.
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.
doi:10.1186/1471-2334-13-432
PMCID: PMC3848720  PMID: 24034230
Multidrug-resistant tuberculosis; Minimum inhibitory concentration; Aminoglycosides; Flouroquinolones; Para-aminosalicylic acid; Ethionamide; rpoB; inhA; embB; pncA
9.  Efficacy of Antiamebic Drugs in a Mouse Model 
Nitroimidazole antibiotics are the mainstay of treatment of invasive amebiasis; however, few comparative studies of applicable antibiotics are available. Evidence of sporadic clinical failure and rare reports of metronidazole resistance have led to the investigation of novel antiamebic therapeutics. The goal of this study was to examine drug efficacy in both in vitro and in vivo models of intestinal amebiasis. We studied six current and three novel drugs. Many drugs, including metronidazole, nitazoxanide, and nitazoxanide derivatives, were shown to be potently inhibitory in vitro. However, metronidazole remained the most effective in vivo, both in preventative and curative regimens, underscoring the value of animal models in evaluating future therapies.
doi:10.4269/ajtmh.2011.10-0580
PMCID: PMC3062453  PMID: 21460014
10.  Diagnosis and Interim Treatment Outcomes from the First Cohort of Multidrug-Resistant Tuberculosis Patients in Tanzania 
PLoS ONE  2013;8(5):e62034.
Setting
Kibong’oto National Tuberculosis Hospital (KNTH), Kilimanjaro, Tanzania.
Objective
Characterize the diagnostic process and interim treatment outcomes from patients treated for multidrug-resistant tuberculosis (MDR-TB) in Tanzania.
Design
A retrospective cohort study was performed among all patients treated at KNTH for pulmonary MDR-TB between November 2009 and September 2011.
Results
Sixty-one culture-positive MDR-TB patients initiated therapy, 60 (98%) with a prior history of TB treatment. Forty-one (67%) were male and 9 (14%) were HIV infected with a mean CD4 count of 424 (±106) cells/µl. The median time from specimen collection to MDR-TB diagnosis and from diagnosis to initiation of MDR-TB treatment was 138 days (IQR 101–159) and 131 days (IQR 32–233), respectively. Following treatment initiation four (7%) patients died (all HIV negative), 3 (5%) defaulted, and the remaining 54 (89%) completed the intensive phase. Most adverse drug reactions were mild to moderate and did not require discontinuation of treatment. Median time to culture conversion was 2 months (IQR 1–3) and did not vary by HIV status. In 28 isolates available for additional second-line drug susceptibility testing, fluoroquinolone, aminoglycoside and para-aminosalicylic acid resistance was rare yet ethionamide resistance was present in 9 (32%).
Conclusion
The majority of MDR-TB patients from this cohort had survived a prolonged referral process, had multiple episodes of prior TB treatment, but did not have advanced AIDS and converted to culture negative early while completing an intensive inpatient regimen without serious adverse event. Further study is required to determine the clinical impact of second-line drug susceptibility testing and the feasibility of alternatives to prolonged hospitalization.
doi:10.1371/journal.pone.0062034
PMCID: PMC3652861  PMID: 23675411
11.  Dual probe DNA capture for sensitive real-time PCR detection of Cryptosporidium and Giardia 
Molecular and Cellular Probes  2011;26(2):104-106.
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 CT 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.
doi:10.1016/j.mcp.2011.12.003
PMCID: PMC3319247  PMID: 22227113
Cryptosporidium; Giardia; PCR; fecal
12.  Molecular Diagnosis of Diarrhea: Current Status and Future Potential 
Determining the microbiologic etiology of enteric infection remains an elusive goal. Conventional approaches, including culture, microscopy, and antigen-based tests have significant limitations such as limit of detection and the need for multiple procedures. Molecular diagnostics, especially PCR based tests, are rapidly changing research and practice in infectious diseases. Diarrheal disease, with its broad range of potential infectious etiologies, is well suited for multiplex molecular testing. This review highlights examples of currently employed molecular tests, as well as ways in which these tests can be applied in the future. The absence of a gold standard for the microbiologic cause of diarrhea means that the clinical significance of detected organisms may not always be clear. Conventional wisdom is that there should be one main pathogen causing diarrhea, however our thinking is challenged by increased detection of mixed infections. Thus, the successful incorporation of molecular diagnostics for diarrheal disease into practice will require both a careful understanding of the technical aspects and research to define their clinical utility.
doi:10.1007/s11908-011-0223-7
PMCID: PMC3253426  PMID: 22116640
diarrhea; molecular diagnosis; PCR; enteropathogens
13.  Protection against Intestinal Amebiasis by a Recombinant Vaccine Is Transferable by T Cells and Mediated by Gamma Interferon▿  
Infection and Immunity  2009;77(9):3909-3918.
We have previously shown that vaccination with purified Entamoeba histolytica Gal/GalNAc lectin or recombinant subunits can protect mice from intestinal amebiasis upon intracecal challenge. In this study, we demonstrated with adoptive-transfer experiments that this lectin vaccine protection is mediated by T cells but not serum. The cell-mediated immune (CMI) response was characterized by significant gamma interferon (IFN-γ), interleukin 12 (IL-12), IL-2, IL-10, and IL-17 production. To move toward a human vaccine, we switched to a recombinant protein and tested a range of adjuvants and routes appropriate for humans. We found that subcutaneous delivery of LecA with IDRI's adjuvant system EM014 elicited a potent Th1-type CMI profile and provided significant protection, as measured by culture negativity (79% efficacy); intranasal immunization with cholera toxin provided 56% efficacy; and alum induced a Th2-type response that protected 62 to 68% of mice. Several antibody and CMI cytokine responses were examined for correlates of protection, and prechallenge IFN-γ+ or IFN-γ-, IL-2-, and tumor necrosis factor alpha-triple-positive CD4 cells in blood were statistically associated with protection. To test the role of IFN-γ in LecA-mediated protection, we neutralized IFN-γ in LecA-immunized mice and found that it abrogated the protection conferred by vaccination. These data demonstrate that CMI is sufficient for vaccine protection from intestinal amebiasis and reveal an important role for IFN-γ, even in the setting of alum.
doi:10.1128/IAI.00487-09
PMCID: PMC2738017  PMID: 19564375
14.  Multiplex PCR method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples 
Cyclospora, Cystoisospora, and Microsporidia are eukaryotic enteropathogens that are difficult to detect in stool samples because they require special stains and microscopy. We developed a multiplex PCR reaction with 4 primer sets to amplify Cyclospora cayetanensis, Cystoisospora belli, Enterocytozoon bieneusi, and Encephalitozoon intestinalis. Detection of the amplicon is through specific probes coupled to Luminex beads. Sensitivity of the assay was evaluated using Encephalitozoon intestinalis spores and revealed detection of 101 spores spiked into stool. No cross reactivity was observed. We evaluated the assay on diarrheal specimens from Thailand, Tanzania, Indonesia, and the Netherlands that had been previously tested by microscopy and the assay yielded 87–100% sensitivity and 88–100% specificity. Microscopy negative/PCR positive samples had lower Luminex values suggesting they were true but lower burden infections. In summary this is a convenient single PCR reaction that can detect Cyclospora, Cystoisospora, and Microsporidia without the need for cumbersome microscopic analysis.
doi:10.1016/j.diagmicrobio.2011.08.012
PMCID: PMC3217099  PMID: 21982218
15.  Disruption of the Human Gut Microbiota following Norovirus Infection 
PLoS ONE  2012;7(10):e48224.
The gut microbiota, the collection of all bacterial members in the intestinal tract, plays a key role in health. Disruption of the indigenous microbiota by a variety of stressors, including antibiotic therapy and intestinal infections, is associated with multiple health problems. We sought to determine if infection with Norovirus disrupts the gut microbiota. Barcoded pyrosequencing of the 16S rRNA-encoding gene was used to characterize the stool microbiota in Norovirus-infected human patients (n = 38). While the microbiota in most infected patients (n = 31) resembled that seen in uninfected healthy controls, a minority of patients (n = 7) possessed a significantly altered microbiota characterized by reduced relative numbers of Bacteriodetes and a corresponding increase in Proteobacteria. In these patients, the increase in Proteobacteria was due to a single operational taxonomic unit (OTU) of Escherichia coli. We cultured E. coli from Norovirus-infected patients and characterized them using PCR-ribotyping and virulence factor analysis. Multiple ribotypes were encountered, but none possessed typical virulence factors commonly carried by enteropathogenic E. coli strains. Microbiota disruption and elevated Proteobacteria were not significantly correlated to patient age, gender, sampling time following illness onset, or overall gut inflammation. These results demonstrate that some patients have a disrupted microbiota following Norovirus infection, and therefore may be at elevated risk for long-term health complications.
doi:10.1371/journal.pone.0048224
PMCID: PMC3484122  PMID: 23118957
16.  High Prevalence of Entamoeba moshkovskii in a Tanzanian HIV population 
Acta tropica  2008;107(1):48-49.
Entamoeba moshkovskii and Entamoeba dispar are microscopically indistinguishable from the pathogenic species Entamoeba histolytica. There are limited data on the prevalence of these commensal infections from Africa. We utilized PCR and antigen detection to evaluate the carriage rate of E. moshkovskii, E. dispar, and E. histolytica infection in stool from a cohort of HIV-suspected or confirmed inpatients from Tanzania. Entamoeba histolytica was detected by ELISA in 4% (5/118) while Entamoeba moshkovskii and E. dispar were detected by PCR in 13% (18/136) and 5% (7/136) of individuals, respectively (P < 0.05). Supporting their commensal nature, neither Entamoeba moshkovskii nor E. dispar infection was statistically associated with HIV status, CD4 count, or the presence of diarrhea. These data suggest E. moshkovskii is a common infection in HIV-infected individuals in northern Tanzania and supports the concept that the microscopic detection of Entamoeba should be interpreted cautiously.
doi:10.1016/j.actatropica.2008.03.013
PMCID: PMC2459240  PMID: 18471796
Entamoeba; moshkovskii; histolytica; dispar; Tanzania; HIV
17.  DETECTION AND QUANTIFICATION OF CRYPTOSPORIDIUM IN HCT-8 CELLS AND HUMAN FECAL SPECIMENS USING REAL-TIME POLYMERASE CHAIN REACTION 
Cryptosporidium is a significant cause of diarrheal illness worldwide, especially among children and immunocompromised patients. Currently used diagnostic techniques are time-consuming, require skilled technicians, and are not useful for quantification of oocysts in fecal and environmental samples. In this study, we examined the use of a real-time polymerase chain reaction (PCR) assay for detecting and quantifying Cryptosporidium parvum in three distinct and progressively more complex matrices: phosphate-buffered saline (PBS), HCT-8 cells (human ileocecal carcinoma), and human fecal specimens. A reliable standard curve was generated using the PBS samples spiked with pure oocysts, and oocyst starting quantities were calculated for the infected HCT-8 cell and spiked fecal samples. The assay detected Cryptosporidium in samples infected/spiked with ≥103 oocysts/sample and detected both C. hominis and C. parvum in clinical specimens. This assay is useful in a variety of samples in the research laboratory and will likely prove to be a useful tool in the clinical laboratory.
PMCID: PMC2253489  PMID: 17488919
18.  Simultaneous Detection of Six Diarrhea-Causing Bacterial Pathogens with an In-House PCR-Luminex Assay 
Journal of Clinical Microbiology  2012;50(1):98-103.
Diarrhea can be caused by a range of pathogens, including several bacteria. Conventional diagnostic methods, such as culture, biochemical tests, and enzyme-linked immunosorbent assay (ELISA), are laborious. We developed a 7-plex PCR-Luminex assay to simultaneously screen for several of the major diarrhea-causing bacteria directly in fecal specimens, including pathogenic Aeromonas, Campylobacter jejuni, Campylobacter coli, Salmonella, Shigella, enteroinvasive Escherichia coli (EIEC), Vibrio, and Yersinia. We included an extrinsic control to verify extraction and amplification. The assay was first validated with reference strains or isolates and exhibited a limit of detection of 103 to 105 CFU/g of stool for each pathogen as well as quantitative detection up to 109 CFU/g. A total of 205 clinical fecal specimens from individuals with diarrhea, previously cultured for enteric pathogens and tested for Campylobacter by ELISA, were evaluated. Using these predicate methods as standards, sensitivities and specificities of the PCR-Luminex assay were 89% and 94% for Aeromonas, 89% and 93% for Campylobacter, 96% and 95% for Salmonella, 94% and 94% for Shigella, 92% and 97% for Vibrio, and 100% and 100% for Yersinia, respectively. All discrepant results were further examined by singleplex real-time PCR assays targeting different gene regions, which revealed 89% (55/62 results) concordance with the PCR-Luminex assay. The fluorescent signals obtained with this approach exhibited a statistically significant correlation with the cycle threshold (CT) values from the cognate real-time PCR assays (P < 0.05). This multiplex PCR-Luminex assay enables sensitive, specific, and quantitative detection of the major bacterial causes of gastroenteritis.
doi:10.1128/JCM.05416-11
PMCID: PMC3256708  PMID: 22075596
19.  Plasma Drug Activity Assay for Treatment Optimization in Tuberculosis Patients ▿ † 
Antimicrobial Agents and Chemotherapy  2011;55(12):5819-5825.
Low antituberculosis (TB) drug levels are common, but their clinical significance remains unclear, and methods of measurement are resource intensive. Subjects initiating treatment for sputum smear-positive pulmonary TB were enrolled from Kibong'oto National TB Hospital, Tanzania, and levels of isoniazid, rifampin, ethambutol, and pyrazinamide were measured at the time of typical peak plasma concentration (C2 h). To evaluate the significance of the effect of observed drug levels on Mycobacterium tuberculosis growth, a plasma TB drug activity (TDA) assay was developed using the Bactec MGIT system. Time to detection of plasma-cocultured M. tuberculosis versus time to detection of control growth was defined as a TDA ratio. TDA assays were later performed using the subject's own M. tuberculosis isolate and C2 h plasma from the Tanzanian cohort and compared to drug levels and clinical outcomes. Sixteen subjects with a mean age of 37.8 years ± 10.7 were enrolled. Fourteen (88%) had C2 h rifampin levels and 11 (69%) had isoniazid levels below 90% of the lower limit of the expected range. Plasma spiked with various concentrations of antituberculosis medications found TDA assay results to be unaffected by ethambutol or pyrazinamide. Yet with a range of isoniazid and rifampin concentrations, TDA exhibited a statistically significant correlation with drug level and drug MIC, and a TDA of ∼1.0 indicated the presence of multidrug-resistant TB. In Tanzania, low (≤2.0) TDA was significantly associated with both lower isoniazid and rifampin C2 h levels, and very low (≤1.5) TDA corresponded to a trend toward lack of cure. Study of TDA compared to additional clinical outcomes and as a therapeutic management tool is warranted.
doi:10.1128/AAC.05561-11
PMCID: PMC3232779  PMID: 21968363
20.  High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites 
Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites—Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis—were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites.
doi:10.4269/ajtmh.2011.10-0461
PMCID: PMC3029193  PMID: 21292910
22.  Comparison of Overnight Pooled and Standard Sputum Collection Method for Patients with Suspected Pulmonary Tuberculosis in Northern Tanzania 
In Tanzania sputum culture for tuberculosis (TB) is resource intensive and available only at zonal facilities. In this study overnight pooled sputum collection technique was compared with standard spot morning collection among pulmonary TB suspects at Kibong'oto National TB Hospital in Tanzania. A spot sputum specimen performed at enrollment, an overnight pooled sputum, and single morning specimen were collected from 50 subjects and analyzed for quality, quantity, and time to detection in Bactec MGIT system. Forty-six (92%) subjects' overnight pooled specimens had a volume ≥5 mls compared to 37 (37%) for the combination of spot and single morning specimens (P < 0.001). Median time to detection was 96 hours (IQR 87–131) for the overnight pooled specimens compared to 110.5 hours (IQR is 137 right 137–180) for the combination of both spot and single morning specimens (P = 0.001). In our setting of limited TB culture capacity, we recommend a single pooled sputum to maximize yield and speed time to diagnosis.
doi:10.1155/2012/128057
PMCID: PMC3335725  PMID: 22567273
23.  Entamoeba histolytica Infection and Secreted Proteins Proteolytically Damage Enteric Neurons ▿  
Infection and Immunity  2010;78(12):5332-5340.
The enteric protozoan parasite Entamoeba histolytica causes amebic colitis through disruption of the mucus layer, followed by binding to and destruction of epithelial cells. However, it is not known whether ameba infections or ameba components can directly affect the enteric nervous system. Analysis of mucosal innervations in the mouse model of cecal amebiasis showed that axon density was diminished to less than 25% of control. To determine whether amebas directly contributed to axon loss, we tested the effect of either E. histolytica secreted products (Eh-SEC) or soluble components (Eh-SOL) to an established coculture model of myenteric neurons, glia, and smooth muscle cells. Neuronal survival and axonal degeneration were measured after 48 h of exposure to graded doses of Eh-SEC or Eh-SOL (10 to 80 μg/ml). The addition of 80 μg of either component/ml decreased the neuron number by 30%, whereas the axon number was decreased by 50%. Cytotoxicity was specific to the neuronal population, since the glial and smooth muscle cell number remained similar to that of the control, and was completely abrogated by prior heat denaturation. Neuronal damage was partially prevented by the cysteine protease inhibitor E-64, showing that a heat-labile protease was involved. E. histolytica lysates derived from amebas deficient in the major secreted protease EhCP5 caused a neurotoxicity similar to that of wild-type amebas. We conclude that E. histolytica infection and ameba protease activity can cause selective damage to enteric neurons.
doi:10.1128/IAI.00699-10
PMCID: PMC2981319  PMID: 20855514
24.  Mycobacterial infections in a large Virginia hospital, 2001-2009 
BMC Infectious Diseases  2011;11:113.
Background
In areas where both tuberculosis (TB) and nontuberculous mycobacteria (NTM) are prevalent, descriptive studies of the clinical features of individual mycobacteria are needed to inform clinical triage.
Methods
We queried the University of Virginia Clinical Data Repository for all mycobacterial infections from 2001-2009.
Results
Of 494 mycobacterial infections in 467 patients there were 22 species. Patients with pulmonary Tb were more likely to be reported as immigrants (p < 0.001) and less likely to have a predisposing risk factor for NTM (pre-existing lung disease or host predisposition; p = 0.002). Review of chest CT scans revealed that TB infection was more likely to exhibit cavities and pleural effusion than NTM infection (p < 0.05). Among NTM infections M. kansasii, M. xenopi, and M. fortuitum were more likely than MAC to have cavities. There were at least 83 patients that met criteria for NTM lung disease and these were caused by 9 species. M. abscessus infection was associated with cystic fibrosis and M. xenopi infection was associated with male gender.
Conclusions
In our center mycobacterial infections were common and of diverse species. Immigrant status, cavities, and effusion were associated with TB vs. NTM.
doi:10.1186/1471-2334-11-113
PMCID: PMC3098778  PMID: 21545738
25.  Therapeutic Drug Monitoring for Slow Response to Tuberculosis Treatment in a State Control Program, Virginia, USA 
Emerging Infectious Diseases  2010;16(10):1546-1553.
TOC summary: Diabetes was associated with increased risk for slow response and low rifampin levels.
Therapeutic drug monitoring may be useful in tuberculosis management, but programmatic implementation is understudied. We performed a retrospective cohort study to determine prevalence of lower than expected levels of isoniazid, rifampin, ethambutol, and pyrazinamide measured at time of estimated peak serum concentration. Patients were tested for serum concentration at 2 hours after medication administration. When patients were tested, 22 had concentrations lower than expected range for rifampin, 23 of 39 patients had low levels of isoniazid, and 8 of 26 patients had low levels of ethambutol; all 20 patients tested for pyrazinamide were within expected range. Over 26 months, 42 patients met criteria for slow response. Diabetes was associated with slow response (p<0.001), and persons with diabetes were more likely than persons without diabetes to have low rifampin levels (p = 0.03). Dosage adjustment of rifampin was more likely to elevate serum concentration to the target range than adjustment of isoniazid given in daily doses (p = 0.01).
doi:10.3201/eid1610.100374
PMCID: PMC3294393  PMID: 20875279
Tuberculosis and other mycobacteria; bacteria; therapeutic drug monitoring; rifampin; isoniazid; diabetes; pharmacokinetics; Virginia; slow response; research

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