Charles P. Cartwright

Diagnostic Performance of Rapid Diagnostic Tests versus Blood Smears for Malaria in US Clinical Practice

BACKGROUND Approximately 4 million US travelers to developing countries are ill enough to seek health care, with 1500 malaria cases reported in the United States annually. The diagnosis of malaria is frequently delayed because of the time required to prepare malaria blood films and lack of technical expertise. An easy, reliable rapid diagnostic test (RDT) with high sensitivity and negative predictive value (NPV), particularly for Plasmodium falciparum, would be clinically useful. The objective of this study was to determine the diagnostic performance of a RDT approved by the US Food and Drug Administration compared with traditional thick and thin blood smears for malaria diagnosis. METHODS This prospective study tested 852 consecutive blood samples that underwent thick and thin smears and blinded malaria RDTs at 3 hospital laboratories during 2003-2006. Polymerase chain reaction verified positive test results and discordant results. RESULTS Malaria was noted in 95 (11%) of the 852 samples. The RDT had superior performance than the standard Giemsa thick blood smear (p = .003). The RDTs sensitivity for all malaria was 97% (92 of 95 samples), compared with 85% (81 of 95) for the blood smear, and the RDT had a superior NPV of 99.6%, compared with 98.2% for the blood smear (p = .001). The P. falciparum performance was excellent, with 100% rapid test sensitivity, compared with only 88% (65 of 74) by blood smear (p = .003). CONCLUSIONS This operational study demonstrates that the US Food and Drug Administration-approved RDT for malaria is superior to a single set of blood smears performed under routine US clinical laboratory conditions. The most valuable clinical role of the RDT is in the rapid diagnosis or the exclusion of P. falciparum malaria, which is particularly useful in outpatient settings when evaluating febrile travelers.

Use of an Enzyme Immunoassay Does Not Eliminate the Need To Analyze Multiple Stool Specimens for Sensitive Detection of Giardia lamblia

ABSTRACT The relative sensitivities of a commercially available enzyme immunoassay (EIA) (ProSpecT Giardia; Alexon-Trend Inc., Ramsey, Minn.) and conventional ovum-and-parasite (O&P) examination for the detection of Giardia lamblia in preserved stool specimens were determined. Paired stool samples collected independently within a 7-day period from 103 patients were analyzed by both methods. A total of 54 specimens from 30 patients (18 asymptomatically infected with G. lamblia and 12 with symptoms consistent with intestinal giardiasis) were determined to be positive for G. lamblia, of which 48 (88.9%) were positive by microscopy and 52 (96.3%) were positive by EIA. Both specimens submitted were positive for G. lamblia by O&P examination for 66.7% (20 of 30) of the positive patients; for 26.7% (8 of 30) a single specimen was positive by O&P examination, and for 6.7% (2 of 30) of those determined to be infected with G. lamblia, both samples were negative by microscopy. The sensitivity of conventional O&P examination was somewhat higher in symptomatically infected individuals, with 75% (9 of 12) of patients in this category havingG. lamblia detected in both samples, compared with 61% (11 of 18) of asymptomatic patients. A total of 24 positive patients (80%) had G. lamblia antigen detected by EIA in both submitted samples, 4 positive patients (13.3%) had one specimen positive by EIA, and the EIA was negative in both specimens from 2 infected individuals (6.5%), the sensitivity of EIA was substantially equivalent in asymptomatic and symptomatic individuals (77 versus 83% of patients with positive results on both specimens). Although the sensitivity of EIA for the detection of G. lamblia on a single stool specimen was somewhat higher than that of conventional O&P examination in symptomatic patients (83 versus 75%), in asymptomatic patients (77 versus 61%), and overall (80 versus 67%), examination of two specimens by either EIA or microscopy was necessary to achieve a diagnostic sensitivity of greater than 90%.

Development and Validation of a Semiquantitative, Multitarget PCR Assay for Diagnosis of Bacterial Vaginosis

ABSTRACT Quantitative PCR assays were developed for 4 organisms reported previously to be useful positive indicators for the diagnosis of bacterial vaginosis (BV)—Atopobium vaginae, Bacterial Vaginosis-Associated Bacterium 2 (BVAB-2), Gardnerella vaginalis, and Megasphaera-1—and a single organism (Lactobacillus crispatus) that has been implicated as a negative indicator for BV. Vaginal samples (n = 169), classified as positive (n = 108) or negative (n = 61) for BV based on a combination of the Nugent Gram stain score and Amsel clinical criteria, were analyzed for the presence and quantity of each of the marker organisms, and the results were used to construct a semiquantitative, multiplex PCR assay for BV based on detection of 3 positive indicator organisms (A. vaginae, BVAB-2, and Megasphaera-1) and classification of samples using a combinatorial scoring system. The prototype BV PCR assay was then used to analyze the 169-member developmental sample set and, in a prospective, blinded manner, an additional 227 BV-classified vaginal samples (110 BV-positive samples and 117 BV-negative samples). The BV PCR assay demonstrated a sensitivity of 96.7% (202/209), a specificity of 92.2% (153/166), a positive predictive value of 94.0%, and a negative predictive value of 95.6%, with 21 samples (5.3%) classified as indeterminate for BV. This assay provides a reproducible and objective means of evaluating critical components of the vaginal microflora in women with signs and symptoms of vaginitis and is comparable in diagnostic accuracy to the conventional gold standard for diagnosis of BV.

Evaluation Of Malaria Screening In Newly Arrived Refugees To The United States By Microscopy And Rapid Antigen Capture Enzyme Assay

Before an empiric malaria treatment program, >60% of Liberian refugees had malaria on arrival to Minnesota. We compared microscopy with rapid antigen testing for detecting asymptomatic parasitemia. Nine of 103 (8.7%) had malaria by polymerase chain reaction (blood smear and rapid testing had a sensitivity of 22%). The empiric treatment program has decreased the rate of imported asymptomatic malaria. Blood film and rapid antigen testing are poor screening tests.

Comparison of Nucleic Acid Amplification Assays with BD Affirm VPIII for Diagnosis of Vaginitis in Symptomatic Women

ABSTRACT A commercially available, nonamplified, nucleic acid probe-based test system (BD Affirm VPIII) was compared with nucleic acid amplification (NAA)-based assays for determining the etiology of vaginitis in a cohort of 323 symptomatic women. First, a semiquantitative, multiplexed PCR assay (BV-PCR) and the Affirm VPIII Gardnerella vaginalis test were compared with a unified bacterial-vaginosis (BV) reference standard incorporating both Nugent Gram stain scores and Amsel clinical criteria. In the evaluable population of 305 patients, BV-PCR was 96.9% (191/197) sensitive and 92.6% specific (100/108) for BV, while Affirm VPIII was 90.1% sensitive (179/197) and 67.6% specific (73/108). Second, a multiplexed PCR assay detecting Candida albicans and Candida glabrata (CAN-PCR) was compared with the Affirm VPIII Candida test using a reference standard for vulvovaginal candidiasis (VVC) of yeast culture plus exclusion of alternate vaginitis etiologies. In the population evaluated (n = 102), CAN-PCR was 97.7% sensitive (42/43) and 93.2% specific (55/59) and Affirm VP III was 58.1% sensitive (25/43) and 100% specific (59/59) for VVC. Finally, the results of a commercial NAA test (GenProbe Aptima Trichomonas vaginalis assay; ATV) for T. vaginalis were compared with the Affirm VPIII Trichomonas vaginalis test. In the absence of an independent reference standard for trichomonal vaginitis (TV), a positive result in either assay was deemed to represent true infection. In the evaluable cohort of 388 patients, the sensitivity of ATV was 98.1% (53/54) versus 46.3% (25/54) for Affirm VPIII. The diagnostic accuracy of the combined NAA-based test construct was approximately 20 to 25% higher than that of the Affirm VPIII when modeled in populations with various prevalences of infectious vaginitis.

Early detection of central nervous system tuberculosis with the gen-probe nucleic Acid amplification assay: utility in an inner city hospital.

Central nervous system tuberculosis is a serious clinical problem, the treatment of which is sometimes hampered by delayed diagnosis. We investigated the utility of the Gen-Probe nucleic acid amplification assay for the rapid diagnosis of tuberculous meningitis and as a noninvasive method of identifying intracranial tuberculoma.

Short communication: Transmitted drug resistance and molecular epidemiology in antiretroviral naive HIV type 1-infected patients in Rhode Island.

Transmission of HIV-1 drug resistance has important clinical and epidemiological consequences including earlier treatment failure and forward transmission of resistance strains in high-risk groups. To evaluate the prevalence and molecular epidemiology of transmitted drug resistance in Rhode Island, we collected genotypic, demographic, clinical, and laboratory data from treatment-naive individuals presenting to the largest outpatient HIV clinic in the state from January 2007 to November 2007. Sequences from 35 treatment-naive individuals were available, 83% of whom were men who had sex with men (MSM). All sequences were HIV-1 subtype B. Drug resistance mutations were identified in 7/35 [20%; 95% confidence interval (CI), 0.08-0.37] patients, six of whom had K103N. Two phylogenetic transmission clusters were found, involving 17% (6/35) of individuals, three in each cluster. We did not find an association between belonging to a cluster and age, gender, AIDS-defining illness, CD4 cell count, or viral load. Drug resistance mutations were more commonly observed in transmission clusters (p = 0.08). Individuals in one cluster all had K103N and were MSM who had attended local bathhouses. Individuals forming clusters were significantly more likely to have visited a bathhouse compared to nonclusters (p = 0.02). The prevalence of transmitted drug resistance in Rhode Island is high, further justifying genotypic testing on presentation to care and prior to treatment initiation. Molecular epidemiological analysis and association of resistance with phylogenetic networks using data obtained for clinical purposes may serve as useful tools for the prevention of drug resistance transmission and for contact tracing.

Use of Sequence Data Generated in the Bayer TruGene Genotyping Assay To Recognize and Characterize Non-Subtype-B Human Immunodeficiency Virus Type 1 Strains

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) gene sequences obtained during antiretroviral resistance testing with a commercial genotyping assay (TruGene; Bayer Corp.) were analyzed to assess the utility of these data for detecting and characterizing non-subtype-B HIV-1 strains. A total of 125 viral sequences obtained from patients believed to have acquired their HIV-1 infection in Africa were analyzed, of which 121 were determined to belong to non-B subtypes. Utilizing TruGene sequence data alone, 92 (76%) of these viruses could be subtyped by conventional phylogenetic analysis. The addition of supplemental RT sequence data enabled a further 28 (23.1%) viruses to be classified, while one (0.9%) sample could not be classified conclusively. Two internet-accessible databases that generate HIV-1 subtypes from PR and RT sequences (HIV-SEQ and Geno2Pheno) were also evaluated, and both achieved 88% concordance (106/120) with phylogenetic analysis. Non-subtype-B and B-subtype HIV-1 sequences could be readily discriminated by tallying silent polymorphisms listed on the TruGene research report. The mean number of silent polymorphisms in the non-B HIV-1 sequences identified in this study was 58.3 (95% confidence interval [CI], 41.1 to 75.5), compared with 20.7 (95% CI, 9.9 to 31.5) for the four subtype B viruses in the study cohort and 118 case-matched B-subtype controls. Sequence data generated in the TruGene HIV-1 genotyping assay could, therefore, provide a ready means of tracking the prevalence and identity of non-B subtypes in HIV-1-infected populations undergoing routine antiretroviral resistance testing.

Evaluation of an Automated Liquid-Handling System (Tecan Genesis RSP 100) in the Abbott LCx Assay for Chlamydia trachomatis

ABSTRACT The present study investigated the feasibility of automating the specimen-pipetting component of sample preparation in the LCx Chlamydia assay (LCx-CT assay; Abbott Laboratories, Chicago, Ill.) by using a commercially available liquid-handling system (Tecan Genesis RSP100; Tecan Inc., Research Triangle Park, N.C.). The Tecan instrument proved to be comparable in both precision and accuracy to a manual multipipettor (Eppendorf model 4850; Eppendorf Scientific, Westbury, N.Y.). The Tecan instrument was extensively checked for evidence of specimen-to-specimen transfer, and no level of contamination sufficient to generate a signal above the background in the LCx-CT assay was detected. Finally, pipetting speed was significantly improved by using the Tecan instrument. A mean time of 2.5 min was required to pipette a complete LCx-CT assay carousel (20 samples and 4 controls) with the Tecan instrument, whereas 8.4 min was required to pipette a comparable number of samples manually (P < 0.001).

Evaluation of Level of Agreement in Bordetella Species Identification in Three U.S. Laboratories during a Period of Increased Pertussis

ABSTRACT While PCR is the most common method used for detecting Bordetella pertussis in the United States, most laboratories use insertion sequence 481 (IS481), which is not specific for B. pertussis; therefore, the relative contribution of other Bordetella species is not understood. The objectives of this study were to evaluate the proportion of other Bordetella species misidentified as B. pertussis during a period of increased pertussis incidence, determine the level of agreement in Bordetella species detection between U.S. commercial laboratories and the CDC, and assess the relative diagnostic sensitivity of CDCs PCR assay when using a different PCR master mix. Specimens collected between May 2012 and May 2013 were tested at two U.S. commercial laboratories for B. pertussis and B. parapertussis detection. Every fifth specimen positive for IS481 and/or IS1001 with cycle threshold (CT ) values of ≤35 was sent to CDC for PCR testing that identifies Bordetella species. Specimens with indeterminate or negative results in the CDC PCR were tested using an alternate PCR master mix. Of 755 specimens, there was agreement in species identification for 83.4% (n = 630). Of the specimens with different identifications (n = 125), 79.2% (n = 99) were identified as indeterminate B. pertussis at CDC. Overall, 0.66% (n = 5) of the specimens were identified as B. holmesii or B. bronchiseptica at CDC. Of 115 specimens with indeterminate or negative results, 46.1% (n = 53) were B. pertussis positive when tested by an alternate master mix, suggesting a possible increase in assay sensitivity. This study demonstrates good agreement between the two U.S. commercial laboratories and CDC and little misidentification of Bordetella species during the 2012 U.S. epidemic.