Chronic desquamative gingivitis (DG) is a common condition often first recognized by periodontists. DG is characterized clinically by erythema with epithelial desquamation, ulceration, and/or the presence of vesiculobullous lesions of the free and attached gingiva. However, DG is actually a clinical manifestation of several different disease processes, particularly lichen planus and benign mucous membrane pemphigoid. Correct diagnosis in DG is critical since proper treatment and follow-up will depend on which disease is involved. However, the diseases that cause DG frequently present diagnostic problems because their lesions often resemble each other clinically and routine histological examination sometimes cannot differentiate between them. Thus, immunohistology, particularly immunofluorescence, is increasingly being used with routine histology to more accurately diagnose DG diseases. This article reviews our experience over the past 10 years in the diagnosis of 72 cases of DG using direct immunofluorescent (DIF) in conjunction with histology and clinical evaluation. Of the 72 DG cases in this study, 30 cases were diagnosed as erosive lichen planus or lichenoid mucositis; 29 cases were diagnosed as benign mucous membrane pemphigoid; 2 cases each of linear IgA disease and pemphigus vulgaris were diagnosed; there was 1 case of bullous pemphigoid; and 1 suspected case of paraneoplastic pemphigus. Even with DIF analysis, 7 cases could not be definitively assigned a particular cause. DIF analysis is not only proving very useful for differential diagnosis, but also adds insight into possible pathogenic mechanisms of DG.

BACKGROUND

Most cases of chronic desquamative gingivitis (CDG) are shown by direct immunofluorescence (DIF) to be immune mediated diseases. Some patients present with similar clinical and microscopic findings as CDG but DIF staining is negative. It has been suggested that those cases of CDG may be hormone (estrogen) mediated and may be treated with estrogens with favorable results.

METHODS

Gingival tissue from 24 cases of CDG and one case of ordinary gingivitis were studied for estrogen receptor (ER) expression using immunohistochemical techniques. Twenty-four of the 25 cases were female. Using standard DIF analysis, 11 of the CDG cases were diagnosed as benign mucous membrane pemphigoid, 10 as lichen planus or lichenoid mucositis (LP), and one as pemphigus. The remaining 3 cases were not diagnostic for a specific disorder (idiopathic). Five of the females had a history of estrogen substitute therapy.

RESULTS

Twenty-two of 23 female CDG cases were positive for ER, although the degree of staining varied. A 32-year-old female with ordinary gingivitis, whose gingivitis varied with her menstrual cycle, did not stain for ER. A 50-year-old male and a 76-year-old female, both with gingival LP, also had negative staining for ER.

CONCLUSIONS

There appears to be no correlation between diagnosed diseases (immunological versus idiopathic) and expression of ER in CDG gingiva. ER expression in the gingiva is probably not related to the presence or absence of estrogen supplementation. The results of this study do not support the use of estrogen in the treatment of idiopathic CDG.

This paper presents an IRT-based statistical test for differential item functioning (DIF). The test is developed for items conforming to the Rasch (Probabilistic models for some intelligence and attainment tests, The Danish Institute of Educational Research, Copenhagen, 1960) model but we will outline its extension to more complex IRT models. Its difference from the existing procedures is that DIF is defined in terms of the relative difficulties of pairs of items and not in terms of the difficulties of individual items. The argument is that the difficulty of an item is not identified from the observations, whereas the relative difficulties are. This leads to a test that is closely related to Lord's (Applications of item response theory to practical testing problems, Erlbaum, Hillsdale, 1980) test for item DIF albeit with a different and more correct interpretation. Illustrations with real and simulated data are provided.

We previously demonstrated that Tribolium castaneum antimicrobical peptide (AMP) genes can be classified to IMD-dependent group I, Toll-dependent group III and co-dependent group II genes besides non-inducible group IV. Here, we focused on NF-κB transcription factor genes, DifDifDif-dependent respectively through knockdown experiments, indicating that the pathway specificity of NF-κB classes found in Drosophila is also conserved in T. castaneum. The Toll-Dif and IMD-Rel pathways of T. castaneum were activated concomitantly by single microbe species, which may represent a distinctive feature of its immune responses. In addition, Rel knockdown impaired host defense against two model bacterial pathogens. Finally, potential κB motifs were searched in the regulatory regions of AMP genes, and relevance to respective NF-κB transcription factors was discussed.

Early and reliable screening for oropharyngeal dysphagia (OD) symptoms in at-risk populations is important and a crucial first stage in effective OD management. The Eating Assessment Tool (EAT-10) is a commonly utilized screening and outcome measure. To date, studies using classic test theory methodologies report good psychometric properties, but the EAT-10 has not been evaluated using item response theory (e.g., Rasch analysis). The aim of this multisite study was to evaluate the internal consistency and structural validity and conduct a preliminary investigation of the cross-cultural validity of the EAT-10; floor and ceiling effects were also checked. Participants involved 636 patients deemed at risk of OD, from outpatient clinics in Spain, Turkey, Sweden, and Italy. The EAT-10 and videofluoroscopic and/or fiberoptic endoscopic evaluation of swallowing were used to confirm OD diagnosis. Patients with esophageal dysphagia were excluded to ensure a homogenous sample. Rasch analysis was used to investigate person and item fit statistics, response scale, dimensionality of the scale, differential item functioning (DIF), and floor and ceiling effect. The results indicate that the EAT-10 has significant weaknesses in structural validity and internal consistency. There are both item redundancy and lack of easy and difficult items. The thresholds of the rating scale categories were disordered and gender, confirmed OD, and language, and comorbid diagnosis showed DIF on a number of items. DIF analysis of language showed preliminary evidence of problems with cross-cultural validation, and the measure showed a clear floor effect. The authors recommend redevelopment of the EAT-10 using Rasch analysis.

OBJECTIVE

Psychological well-being (PWB) has a significant relationship with physical and mental health. As a part of the NIH Toolbox for the Assessment of Neurological and Behavioral Function, we developed self-report item banks and short forms to assess PWB.

METHODS

Expert feedback and literature review informed the selection of PWB concepts and the development of item pools for positive affect, life satisfaction, and meaning and purpose. Items were tested with a community-dwelling US Internet panel sample of adults aged 18 and above (N = 552). Classical and item response theory (IRT) approaches were used to evaluate unidimensionality, fit of items to the overall measure, and calibrations of those items, including differential item function (DIF).

RESULTS

IRT-calibrated item banks were produced for positive affect (34 items), life satisfaction (16 items), and meaning and purpose (18 items). Their psychometric properties were supported based on the results of factor analysis, fit statistics, and DIF evaluation. All banks measured the concepts precisely (reliability ≥0.90) for more than 98% of participants.

CONCLUSIONS

These adult scales and item banks for PWB provide the flexibility, efficiency, and precision necessary to promote future epidemiological, observational, and intervention research on the relationship of PWB with physical and mental health.

BACKGROUND

This study aimed to investigate the reliability and construct validity of the International Classification of Functioning, Disability and Health (ICF) Comprehensive Core Set for osteoarthritis (OA) in order to test its possible use as a measuring tool for functioning.

METHODS

100 patients with OA (84 F, 16 M; mean age 63 yr) completed forms including demographic and clinical information besides the Short Form (36) Health Survey (SF-36®) and the Western Ontario and McMaster Universities Index of Osteoarthritis (WOMAC). The ICF Comprehensive Core Set for OA was filled by health professionals. The internal construct validities of "Body Functions-Body structures" (BF-BS), "Activity" (A), "Participation" (P) and "Environmental Factors" (EF) domains were tested by Rasch analysis and reliability by internal consistency and person separation index (PSI). External construct validity was evaluated by correlating the Rasch transformed scores with SF-36 and WOMAC.

RESULTS

In each scale, some items showing disordered thresholds were rescored, testlets were created to overcome the problem of local dependency and items that did not fit to the Rasch model were deleted. The internal construct validity of the four scales (BF-BS 16 items, A 8 items, P 7 items, EF 13 items) were good [mean item fit (SD) 0.138 (0.921), 0.216 (1.237), 0.759 (0.986) and -0.079 (2.200); person item fit (SD) -0.147 (0.652), -0.241 (0.894), -0.310 (1.187) and -0.491 (1.173) respectively], indicating a single underlying construct for each scale. The scales were free of differential item functioning (DIF) for age, gender, years of education and duration of disease. Reliabilities of the BF-BS, A, P, and EF scales were good with Cronbach's alphas of 0.79, 0.86, 0.88, and 0.83 and PSI's of 0.76, 0.86, 0.87, and 0.71, respectively. Rasch scores of BF-BS, A, and P showed moderate correlations with SF-36 and WOMAC scores where the EF had significant but weak correlations only with SF36-Social Functioning and SF36-Mental Health.

CONCLUSIONS

Since the four different scales derived from BF-BS, A, P, and EF components of the ICF core set for OA were shown to be valid and reliable through a combination of Rasch analysis and classical psychometric methods, these might be used as clinical assessment tools.

The aim of this study was two-fold: (i) to describe the training/match ratios of different external load measures during a full professional soccer season while analyzing the variations between different types of weeks (three, four and five training sessions/week) and (ii) to investigate the relationship between weekly accumulated training loads and the match demands of the same week. Twenty-seven professional soccer players (24.9 ± 3.5 years old) were monitored daily using a 10-Hz global positioning system with a 100-Hz accelerometer. Total distance (TD), running distance (RD), high-speed running (HSR), sprinting distance (SD), player load (PL), number of high accelerations (ACC), and number of high decelerations (DEC) were recorded during training sessions and matches. An individual training/match ratio (TMr) was calculated for each external load measure. Weeks with five training sessions (5dW) presented meaningfully greater TMr than weeks with four (4dW) or three (3dW) training sessions. Additionally, TDratio (TDr) was significantly greater in 5dW than in 3dW (mean differences dif: 1.23 arbitray units A.U.) and 4dW (dif: 0.80 A.U.); HSRr was significantly greater in 5dW than in 3dW (dif: 0.90 A.U.) and 4dW (dif: 0.68 A.U.); and SDr was significantly greater in 5dW than in 3dW (dif: 0.77 A.U.) and 4dW (dif: 0.90 A.U.). Correlations between the weekly training loads and the match demands of the same week were small for PL (r = 0.250 [0.13;0.36]), ACC (r = 0.292 [0.17;0.40]) and DEC (r = 0.236 [0.11;0.35]). This study reveals that ratios of above 1 were observed for specific measures (e.g., HSR, SD). It was also observed that training sessions are not adjusted according to weekly variations in match demands.

Prevalence estimates of depression vary between countries, possibly due to differential functioning of items between settings. This study compared the performance of the widely used Hopkins symptom checklist 15-item depression scale (HSCL-15) across multiple settings using item response theory analyses. Data came from adult populations in the low and middle income countries (LMIC) of Colombia, Indonesia, Kurdistan Iraq, Rwanda, Iraq, Thailand (Burmese refugees), and Uganda (N = 4732).

Item parameters based on a graded response model were compared across LMIC settings. Differential item functioning (DIF) by setting was evaluated using multiple indicators multiple causes (MIMIC) models.

Most items performed well across settings except items related to suicidal ideation and "loss of sexual interest or pleasure," which had low discrimination parameters (suicide: a = 0.31 in Thailand to a = 2.49 in Indonesia; sexual interest: a = 0.74 in Rwanda to a = 1.26 in one region of Kurdistan). Most items showed some degree of DIF, but DIF only impacted aggregate scale-level scores in Indonesia.

Thirteen of the 15 HSCL depression items performed well across diverse settings, with most items showing a strong relationship to the underlying trait of depression. The results support the cross-cultural applicability of most of these depression symptoms across LMIC settings. DIF impacted aggregate depression scores in one setting illustrating a possible source of measurement invariance in prevalence estimates.

The Dictyostelium ras gene, rasD, encodes an mRNA that is more abundant in prestalk than prespore cells in the migratory slug. Its expression is inducible by extracellular cAMP but is not inducible by the prestalk and stalk cell morphogen differentiation inducing factor (DIF). We show that a rasD-lacZ fusion gene is first expressed in approximately one half of the cells in the aggregate, including some cells that also express a prespore-specific marker. The amount of rasD-lacZ fusion protein in prespore cells then diminishes as the slug is formed. Analysis of a rasD-lacZ fusion protein with an N terminal substitution that reduces protein stability within the cell provides strong confirmatory evidence that the ras gene product becomes enriched in prestalk cells by selective repression of gene expression in prespore cells. In contrast, the DIF-inducible ecmA gene is expressed only in those cells that will become prestalk cells in the migratory slug. These results show that there are two different ways in which an mRNA may become enriched in prestalk cells and support the view that DIF is the inducer of prestalk cell differentiation.

DGT (diffusive gradients in thin films) was used to measure the distribution and rates of exchange of Zn, Cd, and Ni between solid phase and solution in five different soils. Soil texture ranged from sandy loam to clay, pH ranged from 4.9 to 7.1, and organic carbon content ranged from 0.8% to 5.8%. DGT devices continuously remove metal to a Chelex gel layer after passage through a well-defined diffusion layer. The magnitude of the induced remobilization flux from the solid phase is related to the pool size of labile metal and the exchange kinetics between dissolved and sorbed metal. DGT devices were deployed over a series of times (4 h to 3 weeks), and the DIFS model (DGT induced fluxes in soils) was used to derive distribution coefficients for labile metal (Kdl) and the rate at which the soil system can supply metal from solid phase to solution, expressed as a response time. Response times for Zn and Cd were short generally (<8 min). They were so short in some soils (<1 min) that no distinction could be made between supply of metal being controlled by diffusion or the rate of release. Generally longer response times for Ni (5-20 min) were consistent with its slow desorption. The major factor influencing Kdl for Zn and Cd was pH, but association with humic substances in the solid phase also appeared to be important. The systematic decline, with increasing pH, in both the pool size of Ni available to the DGT device and the rate constant for its release is consistent with a part of the soil Ni pool being unavailable within a time scale of 1-20 min. This kinetic limitation is likely to limit the availability of Ni to plants.

We examined the relationship of the newly described "Destructive Index" (DI) to emphysema using nine nonemphysematous and 13 emphysematous lungs obtained at autopsy. The amount of emphysema was assessed by the panel method (emphysema grade, EG) and measurement of the mean linear intercept (Lm). The DI depends on three components--alveolar wall/duct disruption, DId; alveolar fibrosis, DIf; and classic emphysema, DIe. DIf was a minor component in our series. The mean DI was 5.8 +/- 2.5, 10.9 +/- 3.9, and 55.7 +/- 7.0% (+/- 1 SEM) in the nonemphysematous (panel grade EG = 0), mild (0 less than EG less than or equal to 25), and moderate to severe (30 less than or equal to EG less than or equal to 60) emphysematous lungs, respectively. The increase in the DI in mild emphysema did not reach significant levels (p less than 0.2). The mean DId was 5.6 +/- 2.5, 10.0 +/- 4.0, and 12.8 +/- 3.9% in the above categories, and the DId in mild emphysema did not differ significantly from that of the nonemphysematous lungs. Lm showed a similar trend and alveolar disruption did not precede airspace enlargement, rather both changes appeared to advance in parallel. The DI correlated closely with EG (r = 0.83, p less than 0.01), but this was due to the component of DIe. The DIe increased steeply in the lungs with EG greater than or equal to 30.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND

When developing new measuring instruments or deciding upon one for research, consideration of the 'best' method of administration for the target population should be made. Current evidence is inconsistent in differentiating superiority of any one method in terms of quantity and quality of response. We trialed a novel mixed methods approach in early scale development to determine the best administration method for a new patient-reported outcome instrument for people with pressure ulcers (the PU-QOL).

METHODS

Cognitive interviews were undertaken with 35 people with pressure ulcers to determine appropriateness of a self-completed version of the PU-QOL instrument. Quantitative analysis, including Rasch analysis, was carried out on PU-QOL data from 70 patients with pressure ulcers, randomised to self-completed or interview-administered groups, to examine data quality and differential item functioning (DIF).

RESULTS

Cognitive interviews identified issues with PU-QOL self-completion. Quantitative analysis supported these findings with a large proportion of self-completed PU-QOLs returned with missing data. DIF analysis indicated administration methods did not impact the way patients from community care settings responded, supporting the equivalence of both administration versions.

CONCLUSIONS

Obtaining the best possible health outcomes data requires use of appropriate methods to ensure high quality data with minimal bias. Mixed methods, with the inclusion of Rasch, provided valuable evidence to support selection of the 'best' administration method for people with PUs during early PRO instrument development. We consider our approach to be generic and widely applicable to other elderly or chronically ill populations or suitable for use in limited samples where recruitment to large field tests is often difficult.

In assessing criminality, researchers have used counts of crimes, arrests, and so on, because interval measures were not available. Additionally, crime seriousness varies depending on demographic factors. This study examined the Crime and Violence Scale (CVS) regarding psychometric quality using item response theory (IRT) and invariance of the crime seriousness hierarchy for gender, age, and racial/ethnic groups on 7,435 respondents. The CVS is a useful measure of criminality, though some items could be improved or dropped. Differential item functioning (DIF) analysis revealed that crime seriousness varies by age and gender. IRT shows promise in assessing and adjusting for demographic variations in crime seriousness.

The collective motion of cells in a biological tissue originates from their individual responses to chemical and mechanical signals. The Dictyostelium slug moves as a collective of up to 100,000 cells with prestalk cells in the anterior 10-30% and prespore cells, intermingled with anterior-like cells (AL cells), in the posterior. We used traction force microscopy to measure the forces exerted by migrating slugs. Wild-type slugs exert frictional forces on their substratum in the direction of motion in their anterior, balanced by motive forces dispersed down their length. StlB- mutants lack the signal molecule DIF-1 and hence a subpopulation of AL cells. They produce little if any motive force in their rear and immediately break up. This argues that AL cells, but not prespore cells, are the motive cells in the posterior zone. Slugs also exert large outward radial forces, which we have analyzed during "looping" movement. Each time the anterior touches down after a loop, the outward forces rapidly develop, approximately normal to the almost stationary contact lines. We postulate that these forces result from the immediate binding of the sheath to the substratum and the subsequent application of outward "pressure," which might be developed in several different ways.

OBJECTIVE

Center for Epidemiologic Studies Depression (CES-D) Scale scores in English- and French-speaking Canadian systemic sclerosis (SSc) patients are commonly pooled in analyses, but no studies have evaluated the metric equivalence of the English and French CES-D. The study objective was to examine the metric equivalence of the CES-D in English- and French-speaking SSc patients.

METHODS

The CES-D was completed by 1007 English-speaking and 248 French-speaking patients from the Canadian Scleroderma Research Group Registry. Confirmatory factor analysis (CFA) was used to assess the factor structure in both samples. The Multiple-Indicator Multiple-Cause (MIMIC) model was utilized to assess differential item functioning (DIF).

RESULTS

A two-factor model (Positive and Negative affect) showed excellent fit in both samples. Statistically significant, but small-magnitude, DIF was found for 3 of 20 CES-D items, including items 3 (Blues), 10 (Fearful), and 11 (Sleep). Prior to accounting for DIF, French-speaking patients had 0.08 of a standard deviation (SD) lower latent scores for the Positive factor (95% confidence interval [CI]-0.25 to 0.08) and 0.09 SD higher scores (95% CI-0.07 to 0.24) for the Negative factor than English-speaking patients. After DIF correction, there was no change on the Positive factor and a non-significant increase of 0.04 SD on the Negative factor for French-speaking patients (difference = 0.13 SD, 95% CI-0.03 to 0.28).

CONCLUSIONS

The English and French versions of the CES-D, despite minor DIF on several items, are substantively equivalent and can be used in studies that combine data from English- and French-speaking Canadian SSc patients.

It has been shown that ammonia inhibits stalk cell formation in monolayers of V12M2, and it was suggested that this inhibition was due to an antagonism of the differentiation-inducing factor (DIF) (Gross, J.D. et al., Nature, 303, 244-246, 1983). However, the results presented here indicate that ammonia inhibition is independent of DIF concentration, and that it occurs well in advance of the period of DIF requirement. Ammonia completely inhibits DIF accumulation and inhibits stalk cell differentiation, but there is no inhibition of prespore cell formation. These results imply the existence of an early ammonia-sensitive event that influences terminal cell type differentiation.

Body checking includes any behavior aimed at global or specific evaluations of appearance characteristics. Men and women are believed to express these behaviors differently, possibly reflecting different socialization. However, there has been no empirical test of the impact of gender on body checking. A total of 1024 male and female college students completed two measures of body checking, the Body Checking Questionnaire and the Male Body Checking Questionnaire. Using multiple group confirmatory factor analysis, differential item functioning (DIF) was explored in a composite of these measures. Two global latent factors were identified (female and male body checking severity), and there were expected gender differences in these factors even after controlling for DIF. Ten items were found to be unbiased by gender and provide a suitable brief measure of body checking for mixed gender research. Practical applications for body checking assessment and theoretical implications are discussed.

To investigate the diagnostic value of a direct immunofluorescence test (DIF-test), urethral samples and first catch urine (FCU) from 153 male patients attending an outpatient clinic for sexually transmitted diseases (STD) were studied. Of the male patients, 40 (26.1%) had a positive urethral culture, 39 (25.5%) had a positive urethral DIF-test, and 32 (20.9%) were positive in urine according to the DIF-test. The sensitivity and the specificity of the DIF-test in male urine specimens were 75% and 98.2%, respectively, as compared with the chlamydial culture, and 69.2% and 95.6%, respectively, as compared to the DIF-test of the urethral samples. Out of the positive urethral samples, 31% had less than 5 elementary bodies (EBs) and 41% greater than 10 EBs, detected by the DIF-test. The corresponding data for FCU were 47% and 22%, indicating a smaller number of chlamydiae in urine than in urethral samples. False negative results in the urine DIF-test were mostly observed in males with a low number of EBs in the urethra. The DIF-test was less sensitive for FCU than for urethral specimens. Therefore, urine DIF-tests cannot replace conventional methods for chlamydial diagnosis in symptomatic STD patients, but may be recommended when genital sampling is not possible and may serve as an important approach in the control of genital chlamydial infections.

In the Dictyostelium slug there are two types of prestalk cells, pstA cells and pstO cells, that differ in their ability to utilize the distal and proximal parts of the promoter of ecmA, a gene that is specifically expressed in prestalk cells. When Rm, a dominant inhibitory form of the regulatory subunit of cAMP-dependent protein kinase (PKA), is expressed under the control of the complete promoter of the ecmA gene (in a construct termed ecmAO:Rm) development proceeds to the slug stage. Although able to form small but outwardly normal slugs, ecmAO:Rm cells are defective in prestalk cell differentiation. In ecmAO:Rm cells, the induction of pstA- and pstO-specific gene expression by the stalk cell inducer DIF is greatly inhibited. Paradoxically, a very large fraction of the cells in an ecmAO:Rm slug show evidence of once having expressed the ecmA and ecmO prestalk markers. However, we present evidence that this is due to abortive prestalk cell differentiation that terminates when sufficient Rm protein has accumulated to block PKA activity. This results in regulative transdifferentiation of prespore cells to form prestalk cells. During their transitory period as prestalk cells the ecmAO:Rm cells coexpress both the ecmA and ecmO markers, indicating a possible link between PKA activity and divergence of the two prestalk cell subtypes. Finally, we show that the level of the DNA binding activity believed to lie at the end of the DIF signal transduction pathway is reduced in ecmAO:Rm slugs.

Subpopulations of patients with adjustment disorder are at increased risk for suicide. The current study investigated whether personality traits, including alexithymia, temperament, and character, are associated with an increased risk of suicide in individuals with adjustment disorder. Age- and sex-matched patients meeting the diagnostic and statistical manual of mental disorders (DSM-IV) criteria for adjustment disorder with (n=92) and without (n=92) a history of suicide attempts were recruited for the present study. Ninety-two healthy individuals who did not meet diagnostic criteria for Axis I or II diagnoses were used as controls. The Toronto alexithymia scale-20 (TAS-20) and the temperament and character inventory (TCI) were used to assess personality traits. Significantly higher total and subscale scores on the TAS-20, including on the difficulty-identifying-feelings (DIF) and difficulty-describing-feelings (DDF) subscales, and lower scores on the TCI cooperativeness subscale were noted in adjustment-disorder patients with previous suicide attempts. In the multivariate regression analysis, high DDF and DIF and low cooperativeness increased the risk of suicide attempts in adjustment-disorder patients. A subsequent path analysis revealed that high DDF had a direct effect on suicide attempts, whereas high DIF had an indirect effect on suicide attempts via low cooperativeness.

The ctxAB operon, encoding cholera toxin (CT) in Vibrio cholerae, is carried by the genome of a filamentous phage, CTXPhi. Usually, specific CTXPhi infect each of the two important biotypes, classical and El Tor, of epidemic V. cholerae strains belonging to serogroup O1, and are called CTX(class)Phi and CTX(ET)Phi, respectively. However, an unusual hybrid El Tor strain carrying CTX(class)Phi caused the cholera epidemic in Mozambique in 2004. To understand the evolution of that strain, we have further analysed some representative hybrid El Tor strains isolated in Kolkata, India, in 1992, and the results indicate that both the Mozambique and the Indian strains are infected with a unique CTX(class)Phi having only four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3' present in the ctxAB promoter (P(ctxAB)) region, like in CTX(ET)Phi. Usually, the P(ctxAB) of the classical biotype contains seven to eight copies of such sequences. However, sequence analyses of the P(ctxAB) regions of several classical strains indicated that the copy number of heptamer repeat sequences might vary from four to eight copies, which was previously unknown. Since the hybrid strains analysed in this study carry four copies of the heptamer sequences, it may thus serve as a marker to trace the strain in future. Interestingly, while the Mozambique strain is devoid of an El Tor-specific free RS1 element or pre-CTX prophage, the Indian hybrid strains carry such elements. The free RS1 has been mapped, cloned and sequenced. As in pre-CTX and CTX prophages, multiple copies of free RS1 elements were found to be integrated in tandem in the large chromosomal dif site. Since Indian hybrid El Tor strains carry either free RS1 or pre-CTX prophage in their large chromosomes, it is possible that the Mozambique hybrid El Tor strain has evolved from these progenitor strains by step-wise deletion of CTX genetic elements from their large chromosomes.