Modelling the potential impact of adherence technologies on tuberculosis in India.

BACKGROUND: For patients taking standard first-line tuberculosis treatment, missing 10% or more of their doses increases the risk of relapse six-fold. Digital technologies offer new approaches to adherence support for TB patients. We estimated the potential impact of new adherence technologies in India.METHOD: We developed a mathematical model of TB transmission dynamics in India, capturing the independent effects of missed doses and treatment default on post-treatment recurrence. We simulated the impact of interventions to address both missed doses and treatment default in the public and private healthcare sector.RESULTS: Adherence interventions, if deployed optimally in the public sector alone, would reduce cumulative TB incidence by 7.3% (95% credible intervals [CrI] 4.7-11) between 2020 and 2030, and by 16% (95% CrI 11-23) if also deployed in the private sector. This impact is roughly proportional to the effectiveness of the interventions. Reducing missed doses reduced incidence by 12% (95% CrI 7.0-18), while reducing treatment default reduced incidence by 7.9% (95% CrI 3.2-13).CONCLUSION: Minimising missed doses is at least as important as promoting treatment completion. Our results suggest that emerging technologies to improve treatment adherence could have a substantial impact on TB incidence and mortality in India.

Tuberculosis infection control measures at health care facilities offering HIV and tuberculosis services in India: A baseline assessment.

BACKGROUND: Tuberculosis (TB) is one of world's oldest infectious disease and ranks alongside HIV as leading infectious killer. Tuberculosis infection control especially in HIV and TB care facilities has warranted attention after the recent health care-associated outbreaks in South Africa. The aim of this study was to describe the tuberculosis infection control measures implemented by HIV and TB care facilities in five high HIV burden provinces in India. METHODS: Baseline assessment of 30 high burden Antiretroviral centers and TB facilities was conducted during Oct 2015-Dec 2015 by AIC trained staff using a structured format. RESULTS: Thirty HIV and TB care facilities in five high HIV burden provinces were enrolled. Facility infrastructure and airborne infection control practices were highly varied between facilities. TB screening and fast tracking at ART centers is happening at majority of centers however inadequate TB infection control training, poor compliance to administrative and personal protective measures and lack of mechanism for health care workers surveillance need attention. CONCLUSIONS: Local specific TB infection control interventions to be designed and implemented at HIV and TB care facilities including implementation of administrative, environmental and use of personal protective equipment's with the training of staff members. Health care workers surveillance needs to be prioritized considering the rising instances of tuberculosis among Health care workers.

Integration and decentralisation of TB-HIV services increases HIV testing of TB cases in Rajasthan, India.

The proportion of tuberculosis (TB) patients tested for the human immunodeficiency virus (HIV) in the state of Ra-jasthan, India, is limited by the availability of HIV testing facilities. Rajasthan implemented a policy of initiating TB-HIV diagnosis at all health institutions in July 2013. The number of TB diagnostic facilities increased from 33 to 63 in Banswara District and from 22 to 68 in Jhunjhunu District, while the number of HIV testing facilities in these districts increased from 1 to 53 and from 10 to 81, respectively, after the policy implementation. The proportion of TB patients tested for HIV increased by respectively 27% and 19%.

La proportion de patients tuberculeux (TB) testés pour le virus de l'immunodéficience humaine (VIH) dans l'état du Rajasthan, Inde, est limitée par la disponibilité de lieux de tests du VIH. Le Rajasthan a mis en œuvre une politique d'initiation du diagnostic TB dans toutes les structures de santé en juillet 2013. Le nombre de lieux de diagnostic TB a augmenté de 33 à 63 dans le district de Banswara et de 22 à 68 dans le district de Jhunjhunu, tandis que le nombre de lieux de test du VIH a augmenté de 1 à 53 et de 10 à 81, respectivement, après la mise en œuvre de cette politique. La proportion de patients TB testés pour le VIH a augmenté de 27% et 19%, respectivement, dans les deux districts.

La proporción de pacientes con tuberculosis (TB) que cuentan con la prueba del virus de la inmunodeficiencia humana (VIH) en el estado de Rajastán en la India se ve limitada por la escasez de establecimientos que la practican. En julio del 2013 se introdujo en Rajastán una política de iniciar el diagnóstico de la TB y el VIH en todos los establecimientos de atención de salud. Después de la introducción de la política, el número de centros de diagnóstico de la TB en el distrito de Banswara aumentó de 33 a 63 y en el distrito Jhunjhunu aumentó de 22 a 68 y la progresión de centros con diagnóstico de la infección por el VIH en Banswazra fue de 1 a 53 centros y en Jhunjhunu de 10 a 81. La proporción de pacientes con TB que cuentan con una prueba del VIH en estos distritos aumentó un 27% y un 19%, respectivamente.

Unfavourable outcomes among patients with MDR-TB on the standard 24-month regimen in Maharashtra, India.

Setting: Patients with multidrug-resistant tuberculosis (MDR-TB) registered for treatment (2011-2012 cohort) using the standard 24-month regimen, under the Revised National TB Control Programme's programmatic management of drug-resistant TB (PMDT), Maharashtra, India. Objectives: To assess the treatment outcomes and the timing and risk factors for unfavourable treatment outcomes, with a focus on death and loss to follow-up (LTFU). Method: This was a retrospective cohort study involving a review of PMDT records. Treatment outcomes were reported on 31 December 2014. Results: Of 4024 patients, treatment success was recorded in 1168 (29%). Unfavourable outcomes occurred in 2242 (56%), of whom 857 (21%) died and 768 (19%) were lost to follow-up. Treatment outcomes were missing on record review for 375 (9%) patients, and 239 (6%) were still undergoing treatment. Half of LTFU occurred within 3 months, and more than four fifths of deaths occurred after 6 months of treatment. Human immunodeficiency virus infection, being underweight, age ⩾ 15 years, male sex and pulmonary TB were the main risk factors for death, LTFU or other unfavourable treatment outcomes. Conclusion: The study found poor treatment outcomes in patients with MDR-TB registered for treatment in Maharashtra, India. Interventions are required to address the high rates of LTFU and death.

Contexte: Les patients atteints d'une tuberculose multi-résistante (TB-MDR) enregistrés en vue d'un traitement (cohorte de décembre 2011) recourant au protocole standard de 24 mois de la prise en charge programmatique de la TB pharmacorésistante (PMDT) sous l'égide du Programme national révisé de lutte contre la TB, à Maharashtra, Inde.Objectifs: Evaluer les résultats du traitement, et le timing et les facteurs de risque de résultats défavorables du traitement en particulier, le décès et les pertes de vue.Méthode: Etude rétrospective de cohorte impliquant la revue des dossiers du PMDT. Les résultats du traitement ont été rapportés au 31 décembre 2014.Résultats: Sur 4024 patients, 1168 (29%) patients ont connu un succès, tandis que 2242 (56%) ont eu un résultat défavorable : 857 (21%) sont décédés et 768 (19%) ont été perdus de vue. Les résultats du traitement ont été manquants lors de la revue des dossiers pour 375 (9%) patients et 239 (6%) patients étaient toujours sous traitement. La moitié des pertes de vue est survenue dans les 3 mois et plus de quatre décès sur cinq sont survenus après 6 mois de traitement. Le virus de l'immunodéficience humaine, la maigreur, l'âge ⩾ 15 ans, le sexe masculin et la TB pulmonaire ont été des facteurs de risque de décès ou de perte de vue ou de résultat défavorable du traitement.Conclusion: Cette étude a découvert des résultats médiocres du traitement chez des patients atteints de TB-MDR enregistrés pour traitement à Maharashtra, Inde. Des interventions sont requises pour combattre ce taux élevé de pertes de vue et de décès.

Marco de referencia: Los pacientes con diagnóstico de tuberculosis multidrogorresistente (TB-MDR) registrados para tratamiento (cohorte de 2011 y 2012) con el régimen normalizado de 24 meses del tratamiento programático de la TB farmacorresistente (PMDT, por su equivalente en inglés) en el marco del Programa Nacional Revisado contra la TB en Maharashtra, en la India.Objetivos: Evaluar los desenlaces terapéuticos, la evolución cronológica y los factores de riesgo de alcanzar un desenlace desfavorable, en especial la muerte y la pérdida durante el seguimiento.Método: Un estudio retrospectivo de cohortes con análisis de los registros del PMDT. Los desenlaces terapéuticos se notificaron el 31 de diciembre del 2014.Resultados: De los 4024 pacientes tratados, 1168 alcanzaron el éxito terapéutico (29%). Se observaron desenlaces desfavorables en 2242 pacientes (56%), así: 857 fallecieron (21%) y 768 se perdieron durante el seguimiento (19%). En el examen de los registros, faltaba el desenlace terapéutico de 375 pacientes (9%) y 239 estaban aun recibiendo tratamiento (6%). La mitad de las pérdidas durante el seguimiento ocurrió durante los primeros 3 meses y más de cuatro quintos de las muertes ocurrieron después de 6 meses de tratamiento. Los factores que se asociaron con la muerte, la pérdida durante el seguimiento u otros desenlaces desfavorables fueron la infección por el virus de la inmunodeficiencia humana, el peso insuficiente, la edad ⩾ 15 años, el sexo masculino y la TB de localización pulmonar.Conclusión: En el presente estudio se observaron desenlaces terapéuticos deficientes en los pacientes registrados en tratamiento por TB-MDR en Maharashtra, en la India. Es necesario introducir intervenciones que aborden la alta tasa de pérdida durante el seguimiento y de muertes.

Bottom-up or top-down: unit cost estimation of tuberculosis diagnostic tests in India.

SETTING: Of 18 sites that participated in an implementation study of the Xpert® MTB/RIF assay in India, we selected five microscopy centres and two reference laboratories. OBJECTIVE: To obtain unit costs of diagnostic tests for tuberculosis (TB) and drug-resistant TB. DESIGN: Laboratories were purposely selected to capture regional variations and different laboratory types. Both bottom-up and the top-down methods were used to estimate unit costs. RESULTS: At the microscopy centres, mean bottom-up unit costs were respectively US$0.83 (range US$0.60-US$1.10) and US$12.29 (US$11.61-US$12.89) for sputum smear microscopy and Xpert. At the reference laboratories, mean unit costs were US$1.69 for the decontamination procedure, US$9.83 for a solid culture, US$11.06 for a liquid culture, US$29.88 for a drug susceptibility test, and US$18.18 for a line-probe assay. Top-down mean unit cost estimates were higher for all tests, and for sputum smear microscopy and Xpert these increased to respectively US$1.51 and US$13.58. The difference between bottom-up and top-down estimates was greatest for tests performed at the reference laboratories. CONCLUSION: These unit costs for TB diagnostics can be used to estimate resource requirements and cost-effectiveness in India, taking into account geographical location, laboratory type and capacity utilisation.

What a difference a day makes: same-day vs. 2-day sputum smear microscopy for diagnosing tuberculosis.

Setting: Nine district-level microscopy centres in Assam and Tripura, India. Objective: Same-day sputum microscopy is now recommended for tuberculosis (TB) diagnosis. We compared this method against the conventional 2-day approach in routine programmatic settings. Methods: During October-December 2012, all adult presumptive TB patients were requested to provide three sputum samples (one at the initial visit, the second 1 h after the first sample, and the third the next morning) for examination by Ziehl-Neelsen smear microscopy. Detection of acid-fast bacilli with any sample was diagnostic. The first and second spot sample comprised the same-day approach, and the first spot sample and next-day sample comprised the 2-day approach. Results: Of 2168 presumptive TB patients, 403 (18.6%) were smear-positive according to the same-day method compared to 427 (19.7%) by the 2-day method (McNemar's test, P < 0.001). Of the total 429 TB patients, 26 (6.1%) were missed by the same-day method and 2 (0.5%) by the 2-day method. Conclusion: Same-day specimen collection for microscopy missed more TB than 2-day collection. In India, missing cases by using same-day microscopy would translate into a considerable absolute number, hindering TB control efforts. We question the indiscriminate switch to same-day diagnosis in settings where patients reliably return for testing the next day.

Contexte : Neuf centres de microscopie de district dans les états d'Assam et de Tripura, Inde.Objectif : On recommande maintenant une microscopie de frottis le même jour pour le diagnostic de la tuberculose (TB). Nous avons comparé cette méthode par rapport à l'approche conventionnelle en 2 jours dans un contexte de programme de routine.Méthodes : Entre octobre et décembre 2012, tous les patients adultes présumés atteints de TB ont été invités à fournir trois échantillons de crachats (lors de la visite initiale, 1 heure après le premier échantillon et le matin suivant) pour un examen par microscopie de frottis selon Ziehl-Neelsen. La détection de bacilles acido-alcoolo-résistants dans un quelconque échantillon constituait le diagnostic. Les premier et deuxième échantillons ont constitué l'approche du même jour et le premier échantillon plus celui du matin suivant constituaient l'approche en 2 jours.Résultats : Sur les 2168 patients présumés TB, 403 (18,6%) étaient frottis-positifs selon la méthode du même jour comparés à 427 (19,7%) par la méthode en 2 jours (test de McNemar P < 0,001). Sur ce total de 429 patients TB, 26 (6,1%) ont été manqués par la méthode du même jour et 2 (0,5%) par la méthode en 2 jours.Conclusion : Le recueil d'échantillons le même jour pour une microscopie a manqué plus de cas de TB que le recueil en 2 jours. En Inde, une stratégie de microscopie le même jour à ce taux se traduirait par un grand nombre absolu de cas manqués, entravant les efforts de lutte contre la TB. Nous remettons en question le fait de passer sans discrimination au diagnostic du jour même dans des contextes où les patients reviennent de manière fiable pour un test le jour suivant.

Marco de referencia: Nueve centros distritales de microscopia de Assam y Tripura en la India.Objetivo: Actualmente se recomienda en el diagnóstico de la tuberculosis (TB) practicar la baciloscopia del esputo el mismo día de la consulta. En el presente estudio se comparó este método con la estrategia corriente de baciloscopia en dos días que se aplica en las prácticas programáticas.Métodos: De octubre a diciembre del 2012 se solicitó a todos los adultos con presunción diagnóstica de TB que aportaran tres muestras de esputo (en el momento de la consulta inicial, una hora después y a la mañana siguiente), con el fin de practicar el examen microscópico con la coloración de Ziehl-Neelsen. El criterio diagnóstico fue la detección de bacilos acidorresistentes en cualquiera de las muestras. La primera y la segunda muestras inmediatas constituyeron la estrategia del mismo día y la primera muestra inmediata y la muestra del día siguiente constituyeron la estrategia de 2 días.Resultados: En los 2168 pacientes con presunción de TB, se confirmó el diagnóstico en 403 casos (18,6%) con el método del mismo día y en 427 (19,7%) con la estrategia de 2 días (prueba de McNemar P < 0,001). Del total de 429 pacientes con diagnóstico de TB, se pasaron por alto 26 casos cuando se recogieron las muestras el mismo día (6,1%) y 2 casos cuando se recogieron durante 2 días (0,5%).Conclusión: La recogida de muestras destinadas a la baciloscopia en el mismo día omitió el diagnóstico de más casos de TB que la recogida de muestras en 2 días. En la India, pasar por alto el diagnóstico de casos con el examen microscópico realizado el mismo día tendría como consecuencia omitir una cifra absoluta de pacientes muy alta, que entorpecería los resultados de las iniciativas de control de la TB. En el presente artículo se pone en duda la utilidad de un cambio indiscriminado hacia el método de diagnóstico en el mismo día, en los entornos donde se puede confiar en que los pacientes regresarán al día siguiente para completar las muestras de esputo.

Device-associated infections at a level-1 trauma centre of a developing nation: impact of automated surveillance, training and feedbacks.

PURPOSE: Device-associated infections constitute the majority of health care-associated infections (HAIs) in ICUs. Trauma patients are predisposed to acquire such infections due to various trauma-related factors. The prevalence of HAIs is underreported from developing nations due to a lack of systematic surveillance. This study reports the impact of an intensive surveillance on the rates and outcome of device-associated infections in trauma patients from a developing country and compares the rates with a previous pilot observation. MATERIALS AND METHODS: The study was conducted at a level-1 trauma centre of India. Surveillance for ventilator-associated pneumonia (VAP), central line-associated blood stream infections (CLA-BSIs) and catheter-associated urinary tract infections (CA-UTIs) was done based on centre for disease control-National Healthcare Safety Network (CDC-NHSN) definitions. The impact of an intensive surveillance, education and awareness drive on the rates of infections over the study period, and compliance to preventive bundles and hand hygiene was assessed. RESULTS: A total of 15,462 ventilator days, 12,207 central line days and 17,740 urinary catheter days were recorded in the study population. The overall rates of VAP, CLA-BSI and CA-UTI were respectively 17, 7.2 and 15.5/1000 device days. There was a significant correlation between device days and the propensity to develop infections. Infections were the cause of death in 36.6% of fatal trauma cases. A significantly higher rate of VAP, CLA-BSI and CA-UTIs was noted in fatal cases. The compliance to ventilator bundle, central line bundle, bladder bundle and hand hygiene were 74.5%, 86%, 79.3% and 64.6%, respectively. A high rate of multi-drug-resistance was observed in all pathogens. A gross reduction in the rates of all infections was observed over time during the study due to implementation of a stringent surveillance system, feedbacks and education. The compliance to hand hygiene and preventive bundles also increased over time. CONCLUSION: The automated surveillance was easy and useful for data entry and analysis. Surveillance had a significant impact on reduction of HAIs and mortality in trauma patients.

Isoniazid preventive treatment in children in two districts of South India: does practice follow policy?

SETTING: Two districts of Tamil Nadu, India OBJECTIVES: To determine the proportion of household contacts aged <6 years of patients with tuberculosis (TB) with positive sputum microscopy results who initiated and completed isoniazid preventive treatment (IPT), and to determine reasons for non-initiation and non-completion of IPT. DESIGN: Household visits were conducted on a random sample of adult patients registered during January-June 2012 to identify household contacts aged <6 years. RESULTS: Among 271 children living with 691 index patients, 218 (80%) were evaluated and 9 (4%) were diagnosed with TB. Of 209 remaining contacts, 70 (33%) started IPT and 16 (22.9%) completed a full course of IPT. Of 139 contacts who did not start IPT, five developed TB disease. Reasons for non-initiation of IPT included no home visit by the field staff (19%) and no education about IPT (61%). Reasons for non-completion included isoniazid not provided (52%) and long duration of treatment (28%). CONCLUSION: This study shows that Revised National TB Programme guidance was not being followed and IPT implementation was poor. Poor IPT uptake represents a missed opportunity to prevent future TB cases. Provision of IPT may be improved through training, improved logistics and enhanced supervision and monitoring.

Same-day light-emitting diode fluorescence microscopy for the diagnosis of tuberculosis in Chhattisgarh, India.

SETTING: Three medical college hospitals using light-emitting diode fluorescence microscopy (LED-FM) for diagnosing tuberculosis (TB) in Chhattisgarh, India. OBJECTIVES: To assess and compare the proportion of sputum smear-positive TB patients diagnosed through same-day microscopy (spot-spot) strategy or with the conventional (spot-morning) strategy. METHODS: During November 2012 - March 2013, all consecutively enrolled presumptive TB patients (aged ≥ 18 years) were requested to provide three specimens: two spot specimens collected 1 h apart on the first day and one early morning specimen the next day; these were stained using auramine-O and examined using LED-FM. RESULTS: Of 1716 (93% of total 1845) presumptive TB patients who provided all three specimens, 218 (13%) were smear-positive: 200 (11.7%) by same-day microscopy and 217 (12.7%) by the conventional method (McNemar's χ(2) 13.5, df 1, P = 0.0002). Eighteen (8.3%) cases were missed by the same-day method. CONCLUSION: Although LED-FM is more sensitive to paucibacillary samples, 8% of smear-positive cases were missed using the same-day method. These findings indicate the need to revisit the global applicability of the current World Health Organization recommendation of switching to same-day diagnosis from the conventional policy.

Composite indicator: new tool for monitoring RNTCP performance in India.

India's Revised National Tuberculosis Control Programme (RNTCP) used the international benchmarks of 70% case detection rate and 85% treatment success rate among new smear-positive tuberculosis (TB) cases for assessing programme performance. This approach overemphasises outcomes and focuses on quantitative benchmarks without sufficient regard to developing systems to monitor appropriate programme practice to achieve a minimum standard of TB care services. The RNTCP has developed a novel composite indicator tool based on a logical framework pathway to move beyond narrow-focused outcome indicators such as case detection to encourage a broad-based analysis of programme implementation. The constituent indicators are from routinely monitored information, spanning input, process, output and outcome indicators across various thematic categories of the RNTCP.

LED fluorescence microscopy increases the detection of smear-positive pulmonary tuberculosis in medical colleges of India.

In July 2012, light-emitting diode fluorescence microscopy (LED-FM) replaced conventional light microscopy using Ziehl-Neelsen stain in the detection of sputum-positive pulmonary tuberculosis in 190 microscopy centres of medical colleges operating under India's Revised National Tuberculosis Control Programme. We compared the performance of LED-FM (July-December 2012) to that of conventional microscopy (July-December 2011) across 190 sites. Of 222 658 patients examined using conventional microscopy, 28 042 (12.6%) were smear-positive, while of 224 714 examined using LED-FM, 33 552 (14.9%) were smear-positive, an additional yield of 5251 cases after adjusting for the increase in patients examined. We recommend replacing conventional microscopy with LED-FM in high workload microscopy centres in India.

Dans 190 centres de microscopie des collèges médicaux travaillant dans le Programme Révisé de Lutte contre la Tuberculose en Inde, l'examen microscopique par fluorescence à diode émettrice de lumière (LED-FM) a remplacé l'examen microscopique à lumière conventionnelle utilisant la coloration de Ziehl-Neelsen pour la détection de la tuberculose pulmonaire à frottis positif. Nous avons comparé les performances du LED-FM entre juillet et décembre 2012 à celles de l'examen microscopique conventionnel (juillet­décembre 2011) dans l'ensemble des 190 sites. Les frottis ont été positifs chez 28 042 (12.6%) des 222 658 patients examinés par microscopie conventionnelle comparé à 33 552 (14.9%) des 224 714 patients examinés par LED-FM, un rendement supplémentaire de 5251 cas après ajustement pour l'augmentation du nombre de patients examinés. Nous recommandons le remplacement de l'examen microscopique conventionnel par le LED-FM en Inde dans les centres de microscopie dont la charge de travail est élevée.

En julio del 2012 la microscopia de fluorescencia con diodos electroluminescentes (LED-FM) reemplazó el sistema tradicional de microscopia de luz con tinción de Ziehl-Neelsen en la detección de casos de tuberculosis pulmonar con baciloscopia positiva en 190 centros de microscopia de las facultadas de medicina que funcionan en conformidad con el Programa Nacional Revisado de Control de la Tuberculosis de la India. En el presente artículo se comparó el rendimiento diagnóstico de este nuevo sistema de microscopia (entre julio y diciembre del 2012) con el rendimiento de la microscopia de luz (entre julio y diciembre del 2011) en 190 centros. De los 222 658 pacientes investigados mediante la microscopia de luz, 28 042 (12,6 %) presentaron baciloscopia positiva. De los 224 714 pacientes cuyas muestras se examinaron mediante la LED-FM, 33 552 (14,9 %) obtuvieron una baciloscopia positiva, lo cual corresponde a un incremento del rendimiento de 5251 casos, después de corregir con respecto al aumento del número de pacientes examinados. Se recomienda sustituir el sistema tradicional de microscopia de luz por la LED-FM en los centros con un alto volumen de trabajo en la India.

Health-care seeking among people with cough of 2 weeks or more in India. Is passive TB case finding sufficient?

SETTING: Thirty districts of India. OBJECTIVES: To estimate 1) the proportion of people with cough of ≥2 weeks, 2) those who did not seek care from a health care provider for cough, and 3) their characteristics. METHODOLOGY: A cross-sectional community-based survey in which 4562 people (aged ≥18 years) were interviewed. RESULTS: Of the 4562 individuals interviewed, 437 (9.5%, 95%CI 7.2-11.8) had cough ≥2 weeks; this was more frequent in those >55 years of age (14%) and in those from districts in eastern (12%) and northern (11%) states of India. Of those with cough, 300 (69%, 95%CI 60-77) had not sought care from any health care provider. Not seeking care was more frequent in people residing in rural areas (73%) compared with urban areas (53%), and in the districts of eastern (82%) and northern (74%) states compared to districts from the southern (46%) and western (54%) states. CONCLUSIONS: Nearly a tenth of those interviewed aged ≥18 years had cough of ≥2 weeks. About two thirds, especially those from rural areas, had not visited a health care provider for the cough. This finding has huge implications for India's current mostly passive case-finding strategy for detecting and controlling tuberculosis.

ras and c-myc oncoproteins during tumor progression in the uterine cervix.

AIMS AND BACKGROUND: Altered oncogenic activity is a feature associated with many malignant and premalignant conditions. Among the many oncogenes, ras and myc are commonly altered in many tumors. This study aims to evaluate the expression of ras and c-myc oncoproteins in a total of 204 cervical tissue samples, including premalignant and malignant lesions as well as apparently normal cervical tissue. METHODS AND STUDY DESIGN: Mouse monoclonal antibodies against the three mammalian ras gene products (c-H-ras, c-K-ras, c-N-ras) and the c-myc protein were used to evaluate oncoprotein expression by immunocytochemistry. RESULTS: None of the samples analyzed displayed immunoreactivity for H-ras and K-ras. Normal cervical epithelium showed minimal immunoreactivity for N-ras with about 33% of the samples expressing the protein. More conspicuous expression in normal tissue was displayed by c-myc, with about 90% of the samples expressing the protein (mean value of cells positive=34%). The immunoreactivity for N-ras increased with increasing histological abnormality from low-grade squamous intraepithelial lesions (SIL) to invasive carcinoma. Increased immunoreactivity for N-ras was evident in the basaloid cells of malignant lesions, with the maximum value of 66% found in poorly differentiated squamous cell carcinoma (PDSCC). The percentage of nuclei positive for c-myc also showed a gradual increase from low-grade SIL onwards, the highest positivity being found in PDSCC, where the mean value was 85%. Statistical analysis revealed a good correlation between the expression of N-ras (r=0.8922, P=0.001) and c-myc (r=0.8856, P=0.001) and various histological stages of tumor progression in the cervical epithelium. CONCLUSIONS: These results therefore suggest that c-myc and N-ras oncoproteins are important during tumor progression in the uterine cervix.

Antichymotrypsin interaction with chymotrypsin. Reactions following encounter complex formation.

Serpins, serine proteinase inhibitors, form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases, that only slowly release I*, in which the P1-P1' linkage is cleaved. Recently we presented evidence that the serpin antichymotrypsin (ACT, I) reacts with the serine proteinase chymotrypsin (Chtr, E) to form an E*I* complex via a three-step mechanism, E + I <==> E .I <==> EI' <==> E*I* in which EI', which retains the P1-P1' linkage, is formed in a partly or largely rate-determining step, depending on temperature (O'Malley, K. H, Nair, S. A., Rubin, H., and Cooperman, B. S. (1997) J. Biol. Chem. 272, 5354-5359). Here we extend these studies through the introduction of a new assay for the formation of the postcomplex fragment, corresponding to ACT residues 359 (the P1' residue) to 398 (the C terminus), coupled with rapid quench flow kinetic analysis. We show that the E.I encounter complex of wild type-rACT and Chtr forms both E*I* and postcomplex fragment with the same rate constant, so that both species arise from EI' conversion to E*I*. These results support our earlier conclusion that the P1-P1' linkage is preserved in EI' and imply that E*I* corresponds to a covalent adduct of E and I, either acyl enzyme or the tetrahedral intermediate formed by water attack on acyl enzyme. Furthermore, we show that the A347R (P12) variant of rACT, which is a substrate rather than an inhibitor of Chtr, has a rate constant for postcomplex fragment formation from the E.I complex very similar to that observed for WT-rACT, implying that EI' is the common intermediate from which partitioning to inhibitor and substrate pathways occurs. These results are used to elaborate a proposed scheme for ACT interaction with Chtr that is considered in the light of relevant results from studies of other serpin-serine proteinase pairs.

The design, synthesis and activity of pentapeptide pp60c-src inhibitors containing L-phosphotyrosine mimics.

Efficient syntheses of 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine and 4-carboxy-L-phenylalanine within the context of the pentapeptide Ac-Ile-X-Gly-Glu-Phe-NH2 (wherein X = the unnatural amino acid) illustrate the use of a divergent synthetic strategy from an advanced common peptide intermediate to more readily access peptide-based tyrosine kinase inhibitors. The key intermediate, Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2, was synthesized by a facile palladium-catalyzed carbonylation of Ac-Ile-Phe(4-iodo)-Gly-Glu(O-tBu)-Phe-NH2. Oxidation of Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2 with tetrabutylammonium permanganate or addition of di-t-butylphosphite, both followed by trifluoroacetic acid deprotection, gave the target pentapeptide inhibitors wherein X = 4-carboxy-L-phenylalanine or 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine, respectively. These two peptides gave somewhat more potent inhibition of the tyrosine kinase pp60c-src than the corresponding pentapeptide wherein X = L-phenylalanine, demonstrating that appended functionalities at the 4-position are accepted and can enhance binding through added interactions within the catalytic region of the active site.

Cytokeratins and the evaluation of tumor differentiation in squamous lesions of the uterine cervix.

The differential expression of cytokeratins in epithelial or squamous cells has been demonstrated to be altered during the process of carcinogenesis. This altered expression of cytokeratins (CKs) may be closely related with epithelial differentiation and may remain stable in malignant tumors. In the present study an analysis using two monoclonal antibodies, CK 8.12 antibody specific for CK type 13 and 16 and CK 8.60 antibody specific for CK type 1, 10 and 11 was done in different grades of lesions in the uterine cervix. Changes from the normal expression pattern were seen in high grade Squamous intraepithelial lesions (SIL) (CIN-2/3) and invasive squamous cell carcinoma (SCC). No conspicuous difference in the staining expression between normal/benign cervical tissue and low grade SIL (CIN-I) was evident. Statistical analysis also revealed a significant correlation between the expression of these CK types to the differentiation status of the cervical lesions analyzed. Alterations in the expression of these CKs can be correlated to the differentiation pathway which may be deregulated during cervical carcinogenesis. The findings of the present study suggest that the expression of CK types 13 and 16 and 1, 10 and 11 using CK 8.12 and CK 8.60 antibodies respectively may serve as markers of differentiation in cervical squamous neoplasms.

The basement membrane and tumor progression in the uterine cervix.

Immunocytochemical localization of the basement membrane (BM) proteins laminin, type-IV collagen and fibronectin were analyzed in normal cervical epithelium, low grade squamous intraepithelial lesions (SILs), high grade SILs and invasive squamous cell carcinoma (SCC) of the uterine cervix. A regular, thick and continuous BM was present in normal cervical epithelium and low grade SIL. Interruptions and discontinuity of the BM were more evident in high grade SILs. There was a good correlation between increasing severity of the lesion and increasing number of breaks. In SCC, the distribution of laminin, collagen IV and fibronectin was related to the degree of cellular differentiation, with decreased immunoreactivity being evident in moderately and poorly differentiated tumors. As the invasive potential of the tumor increased, the fragmentation and loss of BM was more evident. Fibronectin showed only moderate to mild immunoreactivity in normal cervical epithelium and low grade SILs. However, the intensity of expression increased in high grade SILs especially in the peritumoral stroma. It may therefore be concluded from these results that snythesis and reabsorption of BM proteins may be related to shifts in cellular metabolism during tumorigenesis.

The kinetic mechanism of serpin-proteinase complex formation. An intermediate between the michaelis complex and the inhibited complex.

Serine proteinase inhibitors (serpins) form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases that release free enzyme and cleaved inhibitor only very slowly. The mechanism of E*I* formation is incompletely understood and continues to be a source of controversy. Kinetic evidence exists that formation of E*I* proceeds via a Michaelis complex (E.I) and so involves at least two steps. In this paper, we determine the rate of E*I* formation from alpha-chymotrypsin and alpha1-antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of alpha-chymotrypsin with a fluorescent derivative of alpha1-antichymotrypsin (derivatized at position P7 of the reactive center loop); and second, by a rapid mixing/quench approach and SDS-polyacrylamide gel electrophoresis analysis. In some cases, serpins are both substrates and inhibitors of the same enzyme. Our results indicate the presence of an intermediate between E.I and E*I* and suggest that the partitioning step between inhibitor and substrate pathways precedes P1-P1' cleavage.

Increased expression of cytokeratins 14, 18 and 19 correlates with tumor progression in the uterine cervix.

The expression of cytokeratins (CKs) in normal cervical epithelium, low grade squamous intraepithelial lesions (SIL), high grade SILs and squamous cell carcinoma (SCC) were analyzed using four different monoclonal antikeratin antibodies. In normal cervical epithelium, CK 18 showed strong immunoreactivity in basal and parabasal layers. CK 19 and 14 were expressed only in the basal layer while CK 13 was found selectively n the spinal cells. As the lesions progressed from low grade SIL to high grade SIL, immunoreactivity of CK 18, 19 and 14 in the basal cell compartment increased while the expression of CK 13 decreased. In SCC, as well-differentiated tumors showed decreased immunoreactivity for CK 18, 19 and 14 with CK 13 showing a strong and focal (localized) immunoreactivity. Undifferentiated carcinomas totally lacked CK 13 reactivity. Our findings therefore suggest that expression of CK 18, 19 and 14 may be directly related to tumor grade and CK 13 may be a marker of differentiation in cervical lesions.

The effect of epidural fentanyl on the minimum local analgesic concentration of epidural chloroprocaine in labor.

The minimum local analgesic concentration (MLAC) has been defined as the median effective local analgesic concentration (EC50) in a 20-mL volume in the first stage of labor. The aim of this study was to determine the local anesthetic sparing efficacy of epidural fentanyl by its effect on the MLAC of chloroprocaine. Fifty-six parturients, not exceeding 7 cm cervical dilation, who requested epidural analgesia were allocated to one of two groups in this double-blind, randomized, prospective study. After placing a lumbar epidural catheter, 20 mL of the solution being tested was given: chloroprocaine (n = 28) or chloroprocaine with fentanyl 3 micrograms/mL (60 micrograms) (n = 28). The concentration of chloroprocaine was determined by the response of the previous patient to a higher or lower concentration using up-down sequential allocation. Analgesic efficacy was assessed using 100-mm visual analog pain scores with 10 mm or less within 30 min defined as effective. The MLAC of chloroprocaine was reduced from 0.43% wt/vol to 0.26% wt/vol by fentanyl (P = 0.023). Thus, the addition of epidural fentanyl 3 micrograms/mL (60 micrograms resulted in a significant 40% reduction in the MLAC of chloroprocaine in the first stage of labor.