Concordant or discordant results by the tuberculin skin test and the quantiFERON-TB test in children reflect immune biomarker profiles.

The tuberculin skin test (TST) and QuantiFERON-TB-Gold-In-tube (QFTGIT) are adjunctive tests used in the diagnosis of pediatric tuberculosis (TB). Neither test can rule out TB; however, a positive test usually triggers preventive treatment in TB contacts aged <5 years. TST and QFTGIT can give divergent results and it is unclear how discordant results should be interpreted in terms of TB risk and preventive treatment. To understand the immune processes underlying concordant or discordant TST and QFTGIT results, we analyzed immune responses in children from Palamaner Taluk in India (a TB-endemic region with routine neonatal BCG vaccination) who were referred to a TB case verification ward on suspicion of TB. Two hundred and ten children aged <3 years were classified according to their TST and QFTGIT results, and their immune responses analyzed by dual-colour-Reverse-Transcriptase-Multiple-Ligation-dependent-Probe-Amplification, using a panel of 45 genes and a 10-plex antigen-specific enzyme-linked immunosorbent assay. We show that immune biomarkers FPR1, TNFRSF1A and interferon (IFN)-γ are upregulated (all P<0.05) in concordant test-positive children, whereas BPI is downregulated (P<0.05). In contrast, SEC14L1 (P=0.034) and Interferon gamma-induced protein 10 (IP-10) (P=0.001) are differentially expressed between the TST+QFTGIT- /TST-QFTGIT+ groups. Known TB exposure was more frequent in concordant positive children and results were consistent with elevated expression of genes associated with inflammatory responses. Children with discordant test results displayed a mixed profile with activation of both pro- and anti-inflammatory markers. TST and/or QFTGIT positivity appears to reflect distinct but overlapping aspects of host immunity.

Identification of biomarkers for Mycobacterium tuberculosis infection and disease in BCG-vaccinated young children in Southern India.

Pediatric tuberculosis (TB) often goes undiagnosed because of the lack of reliable diagnostic methods. With the aim of assessing biomarker(s) that can aid in the diagnosis of TB infection and disease, we investigated 746 Indian children with suspected TB. Whole-blood mRNA from 210 children was examined by dual-color Reverse-Transcriptase Multiple Ligation-dependent Probe-Amplification for the expression of 45 genes and a Bio-Plex assay for the expression of cytokines/chemokines in QuantiFERON supernatants. The study shows that transcription of SEC14L1, GUSB, BPI, CCR7 and TGFß-1 (all P ≤ 0.05) was downregulated in TB disease compared with uninfected controls, while transcription of RAB33A was downregulated in TB disease compared with both latent TB (P < 0.05) and controls (P < 0.01). The transcription of CD4, TGFß-1 (P < 0.01) and the expression of IL-2 (P < 0.01) and IL-13 (P < 0.05) was upregulated in latent TB compared with that in controls. Using the Least Absolute Shrinkage and Selection Operator (lasso) model, RAB33A alone discriminated between TB disease and latent TB (area under the curve (AUC) 77.5%), whereas a combination of RAB33A, CXCL10, SEC14L1, FOXP3 and TNFRSF1A was effective in discriminating between TB disease and controls (AUC 91.7%). A combination of 11 biomarkers predicted latent TB with moderate discriminatory power (AUC 72.2%). In conclusion, RAB33A is a potential biomarker for TB disease, whereas CD4, TGFß-1 and IL-2, IL-13 may identify latent TB in children.

Mycobacterium tuberculosis Mce1 protein complex initiates rapid induction of transcription of genes involved in substrate trafficking.

The mammalian cell entry (Mce)1 protein complex has an important role during the initial phase of a Mycobacterium tuberculosis (M. tuberculosis) infection. Murine macrophages were infected with M. tuberculosis H37Rv or Δ-mce1 H37Rv, and total RNA was isolated from the host cells at 15, 30 and 60 min, and 4 and 10 h post-infection. With the aim of studying the role for the Mce1 protein complex on host gene expression, the RNA was hybridized onto 44 K whole-genome microarrays. Selected genes were verified by reverse-transcriptase quantitative PCR (RT-QPCR). 'Transport' was the most overrepresented biological process during the first hour post H37Rv infection. Five genes (Abca1 (21.0-fold), Slc16a10 (3.1-fold), Slc6a12 (17.9-fold), Slc6a8 (2.3-fold) and Nr1h3, (5.5-fold)) involved in substrate trafficking were verified by RT-QPCR to be upregulated by >2-fold 1 h post H37Rv infection. By 1 h post Δ-mce1 H37Rv infection, only Abca1 and Slc6a12 were upregulated by >2-fold. A number of other genes, which may be directly involved in substrate trafficking or share the same transcription, were found to have expression profiles similar to the genes involved in substrate trafficking. The Mce1 protein complex has a significant role in the transcriptional activation of genes involved in substrate trafficking during the initial phase of an M. tuberculosis infection.

Rapid syndromic PCR testing in patients with respiratory tract infections reduces time to results and improves microbial yield.

Lack of rapid and comprehensive microbiological diagnosis in patients with community acquired pneumonia (CAP) hampers appropriate antimicrobial therapy. This study evaluates the real-world performance of the BioFire FilmArray Pneumonia panel plus (FAP plus) and explores the feasibility of evaluation in a randomised controlled trial. Patients presenting to hospital with suspected CAP were recruited in a prospective feasibility study. An induced sputum or an endotracheal aspirate was obtained from all participants. The FAP plus turnaround time (TAT) and microbiological yield were compared with standard diagnostic methods (SDs). 96/104 (92%) enrolled patients had a respiratory tract infection (RTI); 72 CAP and 24 other RTIs. Median TAT was shorter for the FAP plus, compared with in-house PCR (2.6 vs 24.1 h, p < 0.001) and sputum cultures (2.6 vs 57.5 h, p < 0.001). The total microbiological yield by the FAP plus was higher compared to SDs (91% (162/179) vs 55% (99/179), p < 0.0001). Haemophilus influenzae, Streptococcus pneumoniae and influenza A virus were the most frequent pathogens. In conclusion, molecular panel testing in adults with CAP was associated with a significant reduction in time to actionable results and increased microbiological yield. The impact on antibiotic use and patient outcome should be assessed in randomised controlled trials.

Direct detection of mycobacterial species in pulmonary specimens by two rapid amplification tests, the gen-probe amplified mycobacterium tuberculosis direct test and the genotype mycobacteria direct test.

Nucleic acid amplification tests have improved tuberculosis diagnostics considerably. This study evaluates a new amplification test, the GenoType Mycobacteria Direct (GTMD) test, for detection of the Mycobacterium tuberculosis complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium malmoense directly in 61 sputum samples. Thirty (49.2%) samples were auramine smear positive, and 31 (50.8%) were smear negative. The GTMD results were compared to the Gen-Probe Amplified M. tuberculosis Direct (MTD) test results, using culturing and sequencing of the 16S rRNA gene as reference methods. The GTMD test could identify 28 of 29 samples containing the M. tuberculosis complex and was negative in a sputum sample containing M. intracellulare. The overall sensitivity and specificity results were 93.3% and 90.0% for the GTMD test, respectively, and 93.1% and 93.5% for the MTD test, respectively. The GTMD test is rapid and can be easily included in routine clinical laboratories for the direct detection of the M. tuberculosis complex in smear-positive sputum samples as an adjunct to microscopy and culture. Further studies are needed to evaluate the performance of the GTMD test for the detection of atypical mycobacteria.

Mucosal antibodies can be measured in air-dried samples of saliva and feces.

IgA antibodies reflecting airways or intestinal mucosal immune responses can be found in saliva and feces, respectively, and IgG antibodies reflecting serum antibodies can be found in saliva. In this study, antibodies were detected in samples of saliva and feces which had been air-dried at room temperature (+20 degrees C) or +37 degrees C, and stored at these temperatures for up to 6 months. In saliva the antibody levels increased, while the antibodies in feces decreased upon storage. The individual IgA antibody concentrations which were adjusted by using the ratios of specific IgA/total IgA were relatively stable in both saliva and feces, and correlated with corresponding antibody levels in samples which had been stored at -20 degrees C. The results indicate that air-dried saliva and feces can be used for semiquantitative measurements of mucosal antibodies, even after prolonged storage at high temperatures and lack of refrigeration.

Measurement of specific IgA in faecal extracts and intestinal lavage fluid for monitoring of mucosal immune responses.

Currently available methods for the evaluation of antigen-specific immune responses in the intestine, i.e. measurement of IgA in intestinal lavage and antibody secreting cells (ASC) in peripheral blood, are not applicable to large-scale immunogenicity studies or to kinetic studies where repeated sampling is required. Simple and reliable methods need to be developed. Intestinal lavage and faecal samples were collected from 12 mice on days 0, 14, 21, 28 and 35 following initial immunization with four doses of cholera toxin (CT) by the gastric or rectal routes. The concentrations of anti-CT IgA in the faecal extracts showed a high level of correlation with those in the lavage samples (Spearman's correlation coefficient=0.85, P<0. 0001) regardless of the route of CT administration. Moreover, the kinetics of the immune response as reflected in the faecal extracts mirrored those in the lavage samples regardless of immunization route. As compared to gastric immunization, rectal administration of CT yielded higher levels of anti-CT IgA in both intestinal lavage fluids and in faecal extracts. The use of rectal immunization and the measurement of IgA in faecal extracts for monitoring mucosal immune responses may be relevant for the development of effective enteric vaccines.

Genotypic detection of enterotoxigenic Escherichia coli colonisation factors.

We have developed a nonradioactive colony hybridisation assay for the detection of enterotoxigenic Escherichia coli (ETEC) that harbor the structural genes for CFA/I, CS1, CS2, CS4, CS17, or PCFO166. Thus, a polynucleotide probe derived from the colonisation factor antigen I (CFA/I) operon hybridised under very low stringency conditions to total DNA from CFA/I-producing (CFA/I), coli-surface antigen 1 and 3 (CS1 CS3-), CS2 CS3-, CS4 CS6-, CS17-, and putative colonisation factor O166 (PCFO166)-producing enterotoxigenic Escherichia coli (ETEC). The probe did not hybridise to DNA from CS3, CFA/III CS6, CS5 CS6, CS6, CS7, or PCFO159 ETEC. Visual registration of colour intensity could be used to differentiate between CFA/I, CS4 and PCFO166-positive strains on the one hand and strains with the genetic potential to express CS1, CS2, or CS17 on the other. As a confirmatory test, restriction fragment patterns obtained from Sau3AI-digested ETEC plasmid DNA could be used to distinguish between CFA/I, CS1, CS4, CS17, and PCFO166 ETEC in nonradioactive Southern blot hybridisation. The simultaneous genotypic detection of several ETEC colonisation factors will prove useful in vaccine-oriented studies of ETEC disease.

Expression of colonization factor antigen I fimbriae by enterotoxigenic Escherichia coli; influence of growth conditions and a recombinant positive regulatory gene.

Enterotoxigenic Escherichia coli (ETEC) may spontaneously lose the positive regulatory cfaR gene and thereby the capacity to express colonization factor antigen I (CFA/I). A recombinant plasmid harbouring the cfaR gene was transformed into cfaR-negative mutant ETEC strains. CFA/I expression of wild-type and cfaR-transformed ETEC cultivated in different liquid media was quantified. At 37 degrees C, a high level of CFA/I expression from wild-type and cfaR-transformed strains was observed after growth in CFA broth. Transformation enhanced CFA/I expression only marginally. The transformant cultures showed a considerable variation in CFA/I expression which was paralleled by the proportion of individual bacteria producing CFA/I. This heterogeneity could be explained by a variable tendency to structural CFA/I gene loss among individual cfaR-transformed bacteria.

Effect of micronutrient deficiency on QuantiFERON-TB Gold In-Tube test and tuberculin skin test in diagnosis of childhood intrathoracic tuberculosis.

BACKGROUND/OBJECTIVES: Data on performance of QuantiFERON-TB Gold In-Tube test (QFT) and tuberculin skin test (TST) in children with active tuberculosis from high burden countries in the context of micronutrient deficiency are scarce. The objective of this study was to evaluate the effect of micronutrient deficiency on the performance of TST and QFT in children with intrathoracic tuberculosis. SUBJECTS/METHODS: Children with probable intrathoracic tuberculosis underwent TST, QFT, gastric lavages and induced sputum examination for AFB (Acid-Fast Bacilli) smear and culture. Zinc, copper, ferritin and vitamin D were measured on stored serum samples. The study used cross-sectional data at initiation of anti-tubercular therapy. RESULTS: Three hundred and sixty-two children (median age 115.5 months (interquartile range: 73, 144), 200 (55.3%) girls) were enrolled in the study. Microbiological confirmation of tuberculosis could be obtained in 128 patients. TST and QFT were positive in 337 (93%) and 297 (82%) children, respectively. Performance of both the tests was unaffected by weight-for-age and height-for-age 'z-scores' or by serum copper levels. TST was not affected by serum zinc and ferritin levels. Children with negative QFT results had lower mean serum zinc level (P=0.01) and higher ferritin levels (P=0.007) as compared to those with positive test. Higher proportion of children with positive TST were vitamin D deficient/insufficient (P=0.003). CONCLUSION: Micronutrient status, especially serum levels of zinc, may influence the performance of QFT in children with intrathoracic tuberculosis. Considering the high prevalence of zinc deficiency in developing countries, QFT should be used cautiously for diagnosing tuberculosis.

Role of the QuantiFERON®-TB Gold In-Tube test in the diagnosis of intrathoracic childhood tuberculosis.

SETTING: Tertiary care hospitals in India. OBJECTIVE: To compare the performance of the QuantiFERON®-TB Gold In-Tube test (QFT-GIT) with that of the tuberculin skin test (TST) in the diagnosis of intrathoracic childhood tuberculosis (TB). METHODS: Children with intrathoracic TB were enrolled in a randomised controlled trial studying micronutrient supplementation in intrathoracic TB. They underwent TST and QFT-GIT before starting daily anti-tuberculosis treatment. RESULTS: Of 362 children (median age 115.5 months, IQR 73-144, 55% girls) enrolled in the study, microbiological confirmation of TB was obtained in 128 (35%). The TST was positive in 337 (93%, 95%CI 90-95.5) and QFT-GIT in 297 (82%, 95%CI 77.8-85.6). Sensitivity of TST and QFT-GIT in culture-confirmed TB cases was respectively 90.5% (95%CI 84.1-94.5) and 82.6% (95%CI 74.9-88.4). QFT-GIT positivity rate correlated with TST induration (P < 0.001). TST was influenced by the disease spectrum (P = 0.004) and the age of the children (P = 0.002); QFT-GIT remained unaffected by these factors. Bacille Calmette-Guérin immunisation status, weight-for-age Z-scores and microbiological confirmation of Mycobacterium tuberculosis did not influence the performance of either test. CONCLUSION: In high-burden countries, QFT-GIT is comparable to TST and offers no added advantage in the diagnosis of childhood intrathoracic TB.

Well-quantified tuberculosis exposure is a reliable surrogate measure of tuberculosis infection.

SETTING: Cape Town, South Africa. OBJECTIVE: To develop a standardized, reliable measure of household tuberculosis (TB) exposure that considers child-specific risk factors. DESIGN: We assessed TB exposure in 536 children. Children were considered Mycobacterium tuberculosis infected if two of three tests of infection were positive. Principal component analysis identified a discrete set of components that collectively described exposure and contributed to a composite contact score. Logistic regression assessed the odds of having M. tuberculosis infection given increasing contact score while controlling for age and past TB treatment. RESULTS: Four components described 68% of data variance: 1) maternal TB and sleep proximity, 2) index case infectivity, 3) duration of exposure, and 4) exposure to multiple index cases. Components were derived from 10 binary questions that contributed to a contact score (range 1-10, median 5, 25th-75th interquartile range [IQR] 4-7). Among children aged 3 months to 6 years with household exposure, the odds of being M. tuberculosis-infected increased by 74% (OR 1.74, 95%CI 1.42-2.12) with each 1-point increase in the contact score. CONCLUSIONS: Well-quantified TB exposure is a good surrogate measure of M. tuberculosis infection in child household contacts in a high-burden setting, and could guide targeted preventive treatment in children at highest risk of M. tuberculosis infection.

Environmental tobacco smoke exposure increases Mycobacterium tuberculosis infection risk in children.

BACKGROUND: Data on the association between exposure to environmental tobacco smoke (ETS) and Mycobacterium tuberculosis infection in children are limited. OBJECTIVE: To examine the dose-response effect of ETS exposure on the risk of M. tuberculosis infection in children in a high tuberculosis (TB) burden setting. METHODS: This cross-sectional study included healthy South African children from impoverished urban communities. Data were collected on household ETS and M. tuberculosis exposure, demographics, socio-economic and anthropometric data, M. tuberculosis infection, human immunodeficiency virus and TB disease status. RESULTS: Among 196 children (median age 6.8 years, range 0.3-15.9), 97 (49.5%) were M. tuberculosis - i nfected (tuberculin skin test [TST] ≥ 10 mm) and 128 (65.3%) reported ETS exposure; of these, 81/128 (63.3%) were exposed to ≥ 2 household smokers. The presence of ≥ 2 household smokers was associated with M. tuberculosis infection in univariate analysis, irrespective of TST cut-off point. In analysis adjusting for M. tuberculosis exposure, socio-economic status, age and previous TB treatment, ETS exposure remained associated with M. tuberculosis infection. In univariate and multivariate analysis, pack-years of exposure were associated with risk of TB infection. DISCUSSION: Exposure to ETS is associated with M. tuberculosis infection in children after adjustment for multiple variables, with a dose-response relationship between the degree of ETS exposure and risk of infection. Public health interventions to reduce exposure to tobacco smoke among children in high TB burden settings are urgently needed.

Short-term reproducibility of a commercial interferon gamma release assay.

Interferon gamma release assays (IGRAs) have been shown to be sensitive and highly specific for the detection of immune memory against Mycobacterium tuberculosis. Little is known about the reproducibility and within-person variability of these assays. Various aspects of short-term reproducibility of a commercial IGRA, the QuantiFERON-TB Gold In-Tube (QFT-IT) assay, were assessed. The QFT-IT assay was performed twice within 3 days in 27 health care workers in Cape Town, South Africa. Two sets of tests were performed by different operators on day 1, and one set was performed on day 3. Aspects such as interoperator, intraoperator, day-to-day variability, and test-retest variability as well as different the storage methods of plasma were investigated. Seventeen of 27 (63%) of participants had at least one positive QFT-IT text; six had discordant results. The agreement of all aspects studied was high, with kappa values between 0.82 and 1.00 for dichotomous measures, and interclass correlations (ICC) of 0.809 to 0.965 were observed for continuous gamma interferon (IFN-gamma) measures. The variability of the magnitude of response was highest comparing measures obtained from individuals on different days (ICC of 0.809). The magnitude of the IFN-gamma responses between assays performed for individual participants was variable, with ranges from 0.03 to 11 IU/ml, resulting is discordant results for five participants. The results indicate that the QFT-IT assay is a robust and highly reproducible assay. Considerable intraindividual variability occurs in the magnitude of IFN-gamma responses, which may influence the interpretation of serial measures.

Evaluation of the nitrate-based colorimetric method for testing the susceptibility of Mycobacterium tuberculosis to streptomycin and ethambutol in liquid cultures.

OBJECTIVES: To evaluate the inexpensive colorimetric nitrate reductase-based antibiotic susceptibility (CONRAS) assay for testing the susceptibility of Mycobacterium tuberculosis to streptomycin and ethambutol in liquid cultures, and to compare the CONRAS test with the manual mycobacteria growth indicator tube (MGIT) test, using the radiometric BACTEC 460TB method as reference. METHODS: A total of 89 M. tuberculosis isolates were tested for susceptibility to streptomycin and ethambutol using the CONRAS and manual MGIT methods and the results were compared with BACTEC 460TB. Isolates with discrepant results between the CONRAS test and BACTEC 460TB were analysed using the agar proportion method, Etest and mutation analysis of genes involved in resistance to streptomycin and ethambutol. RESULTS: The agreement between the CONRAS test and BACTEC 460TB was 88% for streptomycin and 84% for ethambutol. The corresponding agreement of the manual MGIT test with BACTEC 460TB was 89 and 80%, respectively. There was good agreement for streptomycin and moderate agreement for ethambutol between the CONRAS and manual MGIT tests on one hand and BACTEC 460TB on the other (CONRAS test, kappa(streptomycin) 0.74 and kappa(ethambutol) 0.59, P < 0.001; manual MGIT test, kappa(streptomycin) 0.77 and kappa(ethambutol) 0.50, P < 0.001). CONCLUSIONS: There is good agreement for the two non-radiometric liquid culture methods (CONRAS and manual MGIT) compared with BACTEC 460TB for the detection of streptomycin resistance. Further standardization is needed for testing of ethambutol resistance using the CONRAS and manual MGIT assays.

Tuberculosis in contacts need not indicate disease transmission.

BACKGROUND: Traditional contact investigation is an important tool for controlling tuberculosis. It may also help to indicate drug susceptibility patterns when Mycobacterium tuberculosis cultures are not available. Such investigations often underestimate the degree of transmission found by genotyping, but overestimation may also occur. This report is the result of a routine successive DNA restriction fragment length polymorphism (RFLP) analysis of M tuberculosis isolated in Norway. METHOD: Fifteen immigrants belonging to the same community were notified with tuberculosis during February to September 2003. The mycobacterial isolates were analysed by RFLP. RESULTS: All 15 patients had social contact with each other and 13 belonged to the same church community. A total of 14 cultures were positive for M tuberculosis. Among these isolates, six different genotypes were found. Five patients had not acquired the infection from the putative source. CONCLUSIONS: Reactivation of tuberculosis may occur in contacts during the development of an outbreak. In such situations, traditional contact investigations may overestimate the rate of transmission found by genotyping of M tuberculosis. When cultures are unavailable and presumed drug susceptibility patterns are based on that of contacts, such overestimation may lead to incorrect treatment of a patient. Contact investigations must be combined with genotyping of M tuberculosis to conclude how tuberculosis is transmitted. This is especially important in persons with several risk factors for infection.

Simplified and accurate nonradioactive polynucleotide gene probe assay for identification of enterotoxigenic Escherichia coli.

The present study describes a colony hybridization setup for identification of enterotoxigenic Escherichia coli obviating the need for advanced equipment and radioactive isotopes. With a modest laboratory arrangement, polynucleotide gene probes were produced in large quantities. The probes were labeled with digoxigenin and, after hybridization, detected with an antidigoxigenin alkaline phosphatase conjugate. With an established isotope-based oligonucleotide hybridization assay as reference, a blinded study on a large battery of enterotoxigenic and nonenterotoxigenic bacteria revealed a satisfactory sensitivity and specificity of the nonradioactive assay.

Detection and characterization of the coli surface antigen 6 of enterotoxigenic Escherichia coli strains by using monoclonal antibodies.

We describe, for the first time, the production of monoclonal antibodies (MAbs) against coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) and their use for characterization and diagnosis of CS6. Two MAbs, MAbs CS6-20:11:9 and CS6-2A:14, were produced by immunizing mice with purified CS6 or CS6-containing bacterial extracts. The MAb specificity was demonstrated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunoelectron microscopy, which showed that the MAbs bound to CS6-expressing bacteria as well as to purified CS6 and CS6 structural subunits but not to CS6-negative bacteria or other purified ETEC colonization factors. By using bacterial recombinants, i.e., strains with a complete CS6 operon or parts thereof, it was found that both MAbs were specific for CssB, one of the two structural subunits of CS6. Although the MAbs bound specifically to the entire surface of CS6-expressing bacteria, no structure of CS6 could be identified. The usefulness of the MAbs for the detection of CS6 was evaluated in an inhibition ELISA and in a dot blot test. Ninety-two ETEC strains with known colonization factors were analyzed, and all CS6-positive strains were identified by either assay with MAb CS6-2A:14, whereas MAb CS6-20:11:9 failed to identify two CS6-positive strains; in no instance was any CS6-negative strain identified by either of the MAbs. Parallel analyses of 48 strains with a gene probe specific for the other structural subunit of CS6, i.e., CssA, and the MAb-based assays gave identical results, suggesting the simultaneous presence of both subunits.

Use of nonradioactive DNA hybridization for identification of enterotoxigenic Escherichia coli harboring genes for colonization factor antigen I, coli surface antigen 4, or putative colonization factor O166.

We developed an accurate nonradioactive colony hybridization assay (NCHA) using a digoxigenin-labeled polynucleotide probe and an antidigoxigenin alkaline phosphatase conjugate for the identification of enterotoxigenic Escherichia coli (ETEC) harboring genes for colonization factor antigen I (CFA/I), coli surface antigen 4 (CS4), or putative colonization factor O166 (PCFO166). In this 2-day assay, visual registration of color intensity could be used to distinguish between CFA/I-positive strains and strains with the genetic potential to express CS4 or PCFO166. A rapid NCHA was developed by which the results could be read visually 7 h and 45 min after inoculation of the bacteria. In the rapid NCHA, densitometry verified the visual discrimination between four groups of E. coli; ETEC with the CFA/I gene, ETEC with the CS4 gene, ETEC with the PCFO166 gene, and E. coli strains that lack such genes. As a confirmatory test, plasmids from ETEC with the CFA/I, CS4, or PCFO166 gene were differentiated by their characteristic restriction fragment patterns in nonradioactive Southern blot hybridization.