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Tuberculosis (TB) continues to be a serious threat to public health throughout the world. Newer treatments are needed that could offer simplified regimens with activity against both drug‐sensitive and drug‐resistant bacilli, while optimizing safety. Pretomanid (PA‐824), a nitroimidazooxazine compound, is a new drug for the treatment of pulmonary TB that was recently approved in the United States and Europe in the context of a regimen combined with bedaquiline and linezolid. This phase 1 double‐blind, randomized, placebo‐controlled crossover study specifically examined the effect of single‐dose administration of pretomanid 400 or 1000 mg and pretomanid 400 mg plus moxifloxacin 400 mg on the QTc interval in 74 healthy subjects. Subjects were fasting at the time of drug administration. Pretomanid concentrations following single 400‐ or 1000‐mg doses were not associated with any QT interval prolongation of clinical concern. Moxifloxacin did not alter the pharmacokinetics of pretomanid, and the effect of pretomanid 400 mg plus moxifloxacin 400 mg on the individually corrected QT interval was consistent with the effect of moxifloxacin alone. Both drugs were generally well tolerated. Although supratherapeutic exposure of pretomanid relative to the now‐recommended dosing with food was not achieved, these findings contribute to the favorable assessment of cardiac safety for pretomanid.

Objectives: Syria’s protracted conflict has resulted in ideal conditions for the transmission of tuberculosis (TB) and the cultivation of drug-resistant strains. This paper compares TB control in Syria before and after the conflict using available data, examines the barriers posed by protracted conflict and those specific to Syria, and discusses what measures can be taken to address the control of TB in Syria. Results: Forced mass displacement and systematic violations of humanitarian law have resulted in overcrowding and the destruction of key infrastructure, leading to an increased risk of both drug-sensitive and resistant TB, while restricting the ability to diagnose, trace contacts, treat, and follow-up. Pre-conflict, TB in Syria was officially reported at 22 per 100 000 population; the official figure for 2017 of 19 per 100 000 is likely a vast underestimate given the challenges and barriers to case detection. Limited diagnostics also affect the diagnosis of multidrug- and rifampicin-resistant TB, reported as comprising 8.8% of new diagnoses in 2017. Conclusions: The control of TB in Syria requires a multipronged, tailored, and pragmatic approach to improve timely diagnosis, increase detection, stop transmission, and mitigate the risk of drug resistance. Solutions must also consider vulnerable populations such as imprisoned and besieged communities where the risk of drug resistance is particularly high, and must recognize the limitations of national programming. Strengthening capacity to control TB in Syria with particular attention to these factors will positively impact other parallel conditions; this is key as attention turns to post-conflict reconstruction. Keywords: Tuberculosis, Syria, Conflict, Refugees, Drug resistance, Prisoners, Besiegement

All genomes mutate but the consequences of the resulting deleterious mutational load are poorly understood. Helicobacter pylori lives in the human stomach, has a higher mutation rate than most bacteria and has accompanied anatomically modern humans in migrations including the out-of-Africa expansion more than 50,000 years ago. H. pylori from East Asia have accumulated at least 500 more non-synonymous mutations than African strains, which we propose is due to reduced efficacy of selection during the out-of-Africa bottleneck. H. pylori from Europe and the Middle East trace a substantially higher fraction of ancestry from modern African populations than the humans that carry them, which we find is due to at least three separate admixture events. African ancestry is elevated at positions in the genome where non-synonymous mutations are at high frequency in Asia. We propose that this is due to replacement of deleterious mutations that accumulated during the bottleneck, with the high overall African ancestry proportion due to clonal expansion of strains of African origin. We use simulations to show that a Muller9s ratchet like effect can lead to long-term segregation of deleterious mutations within bacterial populations after a bottleneck, despite high rates of homologous recombination, but that population fitness can be restored by migration of small numbers of bacteria from non-bottlenecked populations. Our results demonstrate that population bottlenecks can have long-term genomic and demographic consequences, even in species with enormous population sizes.

Medical diagnosis in low-resource settings is confronted by the lack of suitable guidelines, protocols and checklists. Online-accessible procedural documents are difficult to find, might be mistranslated or interpreted and usually do not address the needs of developing countries. Urinalysis, one of the most frequently performed diagnostic examinations worldwide, involves a series of tests aiming to detect particular disorders, such as urinary tract infections, kidney disease and diabetes. In this guideline, we present an alternative approach for clinical laboratories with limited resources to identify common bacterial uropathogens. We propose dividing the identification plan into two levels. The implicated pathogen will first be assigned into a bacterial group, basic identification, against which a suitable panel of antimicrobial agents shall be selected for the antimicrobial susceptibility testing (AST). Characterization of the pathogen to the genus or species level, advanced identification, will then be performed to ensure correct reading of the AST results and determine the epidemiology of clinically significant pathogens. Most of the proposed steps in our guideline are tailored to meet the needs of clinical laboratories in low-resource settings. Such guidelines are needed to strengthen the capacity of regional pathology laboratories and to enhance international initiatives on antimicrobial resistance and health equity.

CRS3123 is a novel small molecule that potently inhibits methionyl-tRNA synthetase of Clostridioides difficile, inhibiting C. difficile toxin production and spore formation. CRS3123 has been evaluated in a multiple-ascending-dose placebo-controlled phase 1 trial., CRS3123 is a novel small molecule that potently inhibits methionyl-tRNA synthetase of Clostridioides difficile, inhibiting C. difficile toxin production and spore formation. CRS3123 has been evaluated in a multiple-ascending-dose placebo-controlled phase 1 trial. Thirty healthy subjects, ages 18 to 45 years, were randomized into three cohorts of 10 subjects each, receiving either 200, 400, or 600 mg of CRS3123 (8 subjects per cohort) or placebo (2 subjects per cohort) by oral administration twice daily for 10 days. CRS3123 was generally safe and well tolerated, with no serious adverse events (SAEs) or severe treatment-emergent adverse events (TEAEs) reported. All subjects completed their assigned treatment and follow-up visits, and there were no trends in systemic, vital sign, or laboratory TEAEs. There were no QTcF interval changes or any clinically significant changes in other electrocardiogram (ECG) intervals or morphology. CRS3123 showed limited but detectable systemic uptake; although absorption increased with increasing dose, the increase was less than dose proportional. Importantly, the bulk of the oral dose was not absorbed, and fecal concentrations were substantially above the MIC90 value of 1 μg/ml at all dosages tested. Subjects receiving either of the two lower doses of CRS3123 exhibited minimal disruption of normal gut microbiota after 10 days of twice-daily dosing. CRS3123 was inactive against important commensal anaerobes, including Bacteroides, bifidobacteria, and commensal clostridia. Microbiome data showed favorable differentiation compared to other CDI therapeutics. These results support further development of CRS3123 as an oral agent for the treatment of CDI. (This study has been registered at Clinicaltrials.gov under identifier NCT02106338.)

Summary Background Five cases of multi-resistant Acinetobacter baumanii (MRA) producing OXA-23 and OXA-51 occurred in a regional burn intensive care unit (BICU). Three were repatriated from other parts of the world (Dubai and Mumbai) and colonized on admission. Despite optimal precautions, two patients acquired MRA. Both had been nursed in the same room. Methods Multi-disciplinary outbreak investigation of MRA in a regional BICU. Findings The mechanism of transfer for the first case is thought to have been contaminated air from theatre activity releasing MRA bacteria into the communal corridor. No MRA patients went to theatre between the first and second acquired cases. The mechanism of transfer for the second case is thought to have been via a shower unit that was decontaminated inadequately between patients. Conclusion In an outbreak where contact precautions and environmental cleaning are optimal, it is important to give careful consideration to other mechanisms of spread. If there is a failure to do this, it is likely that the true causes of transmission will not be addressed and the problem will recur. It is recommended that burn theatres within burn facilities should be designed to operate at negative pressure; this is the opposite of normal operating theatre ventilation. Where showers are used, both the shower head and the hose should be changed after a patient with a resistant organism. The role of non-contact disinfection (e.g. hydrogen peroxide dispersal) should be reconsidered, and constant vigilance should be given to any ‘trojan horse’ item in the room.

Antimicrobial resistance (AMR) in populations experiencing war has yet to be addressed, despite the abundance of contemporary conflicts and the protracted nature of twenty-first century wars, in combination with growing global concern over conflict-associated bacterial pathogens. The example of the Syrian conflict is used to explore the feasibility of using existing global policies on AMR in conditions of extreme conflict. The available literature on AMR and prescribing behaviour in Syria before and since the onset of the conflict in March 2011 was identified. Overall, there is a paucity of rigorous data before and since the onset of conflict in Syria to contextualize the burden of AMR. However, post onset of the conflict, an increasing number of studies conducted in neighbouring countries and Europe have reported AMR in Syrian refugees. High rates of multidrug resistance, particularly Gram-negative organisms, have been noted amongst Syrian refugees when compared with local populations. Conflict impedes many of the safeguards against AMR, creates new drivers, and exacerbates existing ones. Given the apparently high rates of AMR in Syria, in neighbouring countries hosting refugees, and in European countries providing asylum, this requires the World Health Organization and other global health institutions to address the causes, costs, and future considerations of conflict-related AMR as an issue of global governance. (c) 2018 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

Otitis externa is the inflammation of the external auditory canal. The disease is common and shows a seasonal variation with a greater incidence in warmer months. Pseudomonas aeruginosa is a common pathogen in otitis externa and in this retrospective study, we show a corresponding seasonal variation in the proportional incidence of P. aeruginosa isolates from otitis externa in South East England. In total 7770 patients were diagnosed with otitis externa over a period of 9 years from January 2008 to December 2016. P. aeruginosa was isolated from 2802 patients (proportional incidence of 36%). Incidence was higher in the months of August, September and October and in patients between 5 and 15 years of age. We postulate a combination of increased contact with water during warm weather in the holiday season and increased rainfall in the preceding season as a putative mechanism for the seasonal trends.

Current evidence describing antimicrobial resistance (AMR) in the context of the Syrian conflict is of poor quality and sparse in nature. This paper explores and reports the major drivers of AMR that were present in Syria pre-conflict and those that have emerged since its onset in March 2011. Drivers that existed before the conflict included a lack of enforcement of existing legislation to regulate over-the-counter antibiotics and notification of communicable diseases. This contributed to a number of drivers of AMR after the onset of conflict, and these were also compounded by the exodus of trained staff, the increase in overcrowding and unsanitary conditions, the increase in injuries, and economic sanctions limiting the availability of required laboratory medical materials and equipment. Addressing AMR in this context requires pragmatic, multifaceted action at the local, regional, and international levels to detect and manage potentially high rates of multidrug-resistant infections. Priorities are (1) the development of a competent surveillance system for hospital-acquired infections, (2) antimicrobial stewardship, and (3) the creation of cost-effective and implementable infection control policies. However, it is only by addressing the conflict and immediate cessation of the targeting of health facilities that the rehabilitation of the health system, which is key to addressing AMR in this context, can progress.

Studies on candidate pandemic vaccines against avian influenza viruses have focused on H5N1, but viruses of other subtypes, such as A/H9N2, are also considered to have pandemic potential. We investigated the safety and immunogenicity of two immunizations with one of five different antigen doses (ranging from 3.75 to 45 μg of hemagglutinin antigen) of a nonadjuvanted whole-virus G9 lineage H9N2 influenza virus vaccine in healthy adults aged 18 to 49 years. The antibody responses were measured by hemagglutination inhibition (HI), microneutralization (MN), and single radial hemolysis (SRH) assays. To investigate a hypothesis that previous exposure to H2N2 viruses in subjects born in or before 1968 might prime for more robust antibody responses to H9N2 vaccination than that in subjects born after 1968, apost hocage-stratified analysis of antibody responses was done. Both vaccinations in all dose groups were safe and well tolerated. No vaccine-related serious adverse events were reported, and the majority of the adverse reactions were rated as mild. The rates of injection site reactions were lower in the 3.75-μg- and 7.5-μg-dose groups than those in the higher-dose groups; the rates of systemic reactions were similar across all dose groups. The seroprotection rates among the different dose groups 21 days after the second immunization ranged from 52.8% to 88.9% as measured by HI assay, from 88.7% to 98.1% or 82.7% to 96.2% as measured by MN assay (MN titer cutoffs, 1:40 and 1:80, respectively), and from 94.2% to 100% as measured by SRH assay. Higher antibody responses were not induced in subjects born in or before 1968. These data indicate that a nonadjuvanted whole-virus H9N2 vaccine is well tolerated and immunogenic in healthy adults. (This study has been registered atClinicalTrials.govunder registration no. NCT01320696.)

Background Cell culture technologies have the potential to improve the robustness and flexibility of influenza vaccine supply and to substantially shorten manufacturing timelines. We investigated the safety, immunogenicity and efficacy of a Vero cell culture-derived seasonal influenza vaccine and utilized these studies to establish a serological correlate of vaccine protection. Methods Two multicenter, randomized, double-blind phase III trials were undertaken in the US during the 2008–2009 Northern hemisphere influenza season, in young (18–49 years) and older (50–64 years and ≥65 years) adult subjects. 7250 young adults were randomized 1:1 to receive either Vero-derived vaccine or placebo. 3210 older adult subjects were randomized 8:1 to receive either Vero-derived vaccine or a licensed egg-derived vaccine. Serum hemagglutination inhibition antibody titers were assessed 21 days post-vaccination. Vaccine efficacy in preventing cell culture-confirmed influenza infection was determined for the young adult population. Local and systemic adverse events were recorded in both studies. Results The Vero-derived vaccine was safe and well tolerated in both young and older adults. All US and European immunological licensing thresholds were comfortably met in both populations. Vaccine efficacy in young adults was 79% against A/H1N1 viruses antigenically matching the corresponding vaccine strain and 78.5% for all antigenically matched influenza viruses. A hemagglutination inhibition antibody titer of ≥1:15 provided a reliable correlate of protection for the Vero-derived influenza vaccine, with no additional benefit at titers >1:30. Bridging of the correlate of protection established in the young adult population to the older adult immunogenicity data demonstrated the likely effectiveness of the Vero-derived vaccine in the older adult population. Conclusions A Vero cell culture-derived seasonal influenza vaccine is safe, immunogenic and protects against infection with influenza virus. The novel vaccine technology has the potential to make a substantial contribution to improving influenza vaccine supply. Clinical trial registration The studies are registered with ClinicalTrials.gov, numbers NCT00566345 and NCT00782431 .

A Vero cell culture–derived seasonal influenza vaccine provides consistently high levels of protection against cell culture–confirmed infection over a complete influenza season. Influenza symptoms are also less severe and of shorter duration in individuals who become infected despite vaccination., Background. Current knowledge of the consistency of protection induced by seasonal influenza vaccines over the duration of a full influenza season is limited, and little is known about the clinical course of disease in individuals who become infected despite vaccination. Methods. Data from a randomized double-blind placebo-controlled clinical trial undertaken in healthy young adults in the 2008–2009 influenza season were used to investigate the weekly cumulative efficacy of a Vero cell culture–derived influenza vaccine. In addition, the duration and severity of disease in vaccine and placebo recipients with cell culture–confirmed influenza infection were compared. Results. Vaccine efficacy against matching strains was consistently high (73%–82%) throughout the study, including the entire period of the influenza season during which influenza activity was above the epidemic threshold. Vaccine efficacy was also consistent (68%–83%) when calculated for all strains, irrespective of antigenic match. Vaccination also ameliorated disease symptoms when infection was not prevented. Bivariate analysis of duration and severity showed a significant amelioration of myalgia (P = .003), headache (P = .025), and fatigue (P = .013) in infected vaccinated subjects compared with placebo. Cough (P = .143) and oropharyngeal pain (P = .083) were also reduced in infected vaccinated subjects. Conclusions. A Vero cell culture–derived influenza vaccine provides consistently high levels of protection against cell culture–confirmed infection by seasonal influenza virus and significantly reduces the duration and severity of disease in those individuals in which infection is not prevented. Clinical Trials Registration. ClinicalTrials.gov NCT00566345.

Since 2013, wounded and ill children from Syria have received treatment in Israel. Screening cultures indicated that multidrug-resistant (MDR) pathogens colonized 89 (83%) of 107 children. For 58% of MDR infections, the pathogen was similar to that identified during screening. MDR screening of these children is valuable for purposes of isolation and treatment.

Background Ototoxicity is a severe side effect of aminoglycoside antibiotics. Aminoglycosides are recommended for the treatment of multidrug-resistant TB (MDR-TB). N -Acetylcysteine (NAC) appears to protect against drug- and noise-induced hearing loss. This review aimed to determine if coadministering NAC with aminoglycoside affected ototoxicity development, and to assess the safety and tolerability of prolonged NAC administration. Methods Eligible studies reported on the efficacy of concomitant NAC and aminoglycoside administration for ototoxicity prevention or long-term (≥6 weeks) administration of NAC regardless of indication. Pooled estimates were calculated using a fixed-effects model. Heterogeneity was assessed using the I 2 statistic. Results Three studies reported that NAC reduced ototoxicity in 146 patients with end-stage renal failure receiving aminoglycosides. Pooled relative risk for otoprotection at 4–6 weeks was 0.14 (95% CI 0.05 to 0.45), and the risk difference was −33.3% (95% CI 45.5% to 21.2%). Eighty-three studies (N=9988) described the administration of NAC for >6 weeks. Abdominal pain, nausea and vomiting, diarrhoea and arthralgia were increased 1.4–2.2 times. Discussion This review provides evidence for the safety and otoprotective effect of NAC when coadministered with aminoglycoside. It represents a strong justification for a clinical trial to investigate the effect of concomitant NAC treatment in patients receiving aminoglycosides as part of MDR-TB treatment.

Unbiased species-level identification of coagulase-negative staphylococci (CoNS) using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identified Staphylococcus lugdunensis to be a more commonly isolated CoNS in our laboratory than previously observed. It has also highlighted the possibility of vertical transmission.

Background Training in microbiology is continuing to evolve. Standardisation of this process has, in part, been achieved through the development of a training curriculum by the Royal College of Pathologists (RCPath). A substantial proportion of microbiology training occurs through telephone consultations. Aims To ascertain the content of these interactions and the extent to which the necessary skills outlined by the curriculum are attainable via these consultations. Methods Records of telephone consultations made by microbiology registrars (SpR) on the Laboratory Information Management System (LIMS) over a 6 month period were analysed with regard to who initiated contact and the type of advice provided. Results An average of 426 SpR entries per month were made on the LIMS following telephone consultations. These consultations were predominantly initiated by fellow clinicians as opposed to the SpR. The majority (79%) of advice entailed guidance as to the use of antimicrobials which resulted in an alteration of the current regimen in 54% of cases. Conclusions This study represents the first attempt to quantify the telephone consultations of microbiology trainees. It is concluded that although such interactions provide a means of attaining some of the competencies outlined by the RCPath curriculum, the bias towards antimicrobial advice reflects a discrepancy between the needs of the service users and the broad skill set advocated by the current microbiology training programme. Future modifications will need to take this into account to ensure both the training of SpRs and the microbiology service is fit for purpose.

Both anatomically modern humans and the gastric pathogen Helicobacter pylori originated in Africa, and both species have been associated for at least 100,000 years. Seven geographically distinct H. pylori populations exist, three of which are indigenous to Africa: hpAfrica1, hpAfrica2, and hpNEAfrica. The oldest and most divergent population, hpAfrica2, evolved within San hunter-gatherers, who represent one of the deepest branches of the human population tree. Anticipating the presence of ancient H. pylori lineages within all hunter-gatherer populations, we investigated the prevalence and population structure of H. pylori within Baka Pygmies in Cameroon. Gastric biopsies were obtained by esophagogastroduodenoscopy from 77 Baka from two geographically separated populations, and from 101 non-Baka individuals from neighboring agriculturalist populations, and subsequently cultured for H. pylori. Unexpectedly, Baka Pygmies showed a significantly lower H. pylori infection rate (20.8%) than non-Baka (80.2%). We generated multilocus haplotypes for each H. pylori isolate by DNA sequencing, but were not able to identify Baka-specific lineages, and most isolates in our sample were assigned to hpNEAfrica or hpAfrica1. The population hpNEAfrica, a marker for the expansion of the Nilo-Saharan language family, was divided into East African and Central West African subpopulations. Similarly, a new hpAfrica1 subpopulation, identified mainly among Cameroonians, supports eastern and western expansions of Bantu languages. An age-structured transmission model shows that the low H. pylori prevalence among Baka Pygmies is achievable within the timeframe of a few hundred years and suggests that demographic factors such as small population size and unusually low life expectancy can lead to the eradication of H. pylori from individual human populations. The Baka were thus either H. pylori-free or lost their ancient lineages during past demographic fluctuations. Using coalescent simulations and phylogenetic inference, we show that Baka almost certainly acquired their extant H. pylori through secondary contact with their agriculturalist neighbors., Author Summary Genetic analyses of Helicobacter pylori have illuminated human migrations and the history of human infection by these bacteria. Both humans and H. pylori originated in Africa, and have been intimately associated for at least 100,000 years. We hypothesized that communities who still live in relative isolation might provide further details about the evolutionary history of H. pylori in Africa. We therefore investigated H. pylori within Baka Pygmies of southeast Cameroon, who live as hunter-gatherers in the tropical rainforest, and compared those bacteria to H. pylori from neighboring farming populations of non-Baka ethnicities. Unexpectedly, Baka Pygmies were much less commonly infected (20.8%) than the non-Baka (80.2%). H. pylori from hunter-gatherers and agriculturalists were genetically very similar and ancient H. pylori lineages were not identified in Baka. We used an epidemiological model to show that demographic factors including small population size and low life expectancy can account for the low infection rate among Baka Pygmies, and that this low rate could have been attained within a few hundred years of secondary contact with their neighbors. We also suggest that the ancestors of the Baka Pygmies were initially H. pylori-free or that their ancestral bacteria have been lost through past demographic fluctuations.

Summary Background The use of cell-culture technologies for the manufacture of influenza vaccines might contribute to improved strain selection and robust vaccine supplies. We investigated the safety, immunogenicity, and protective efficacy of a Vero-cell-culture-derived influenza vaccine, and assessed the correlation between vaccine efficacy and haemagglutination inhibition antibody titre. Methods In a double-blind, placebo-controlled, phase 3 trial undertaken in 36 centres in the USA, healthy adults (aged 18–49 years) were randomly assigned in a 1:1 ratio to one injection of either placebo or Vero-cell-culture-derived influenza vaccine during the 2008–09 season. Randomisation was done in blocks by use of the random number generator algorithm, and participants were allocated by use of a centralised telephone system. The primary objective was the efficacy of the vaccine in preventing cell-culture-confirmed influenza infection with viruses that were antigenically matched to one of the vaccine strains. Analysis was by intention to treat. The study is registered with ClinicalTrials.gov, number NCT00566345. Findings 7250 participants were randomly assigned to vaccine (n=3626) and placebo (n=3624). 7236 were analysed for the primary outcome (n=3619 and n=3617, respectively). Overall protective efficacy for antigenically matched influenza infection was 78·5% (95% CI 60·8–88·2). The vaccine was well tolerated with no treatment-related serious adverse events. Adverse events were mainly mild and transient. An HI titre of at least 1:15 provided a reliable correlate of cell-culture-derived influenza vaccine-induced protection; no additional benefit was noted with titres greater than 1:30. Interpretation The data indicate that existing correlates of protection afforded with egg-derived seasonal influenza vaccines also apply to this vaccine. Funding Federal (US Government) funds from the Office for Preparedness and Response, Biomedical Advanced Research and Development Authority, under contract to DynPort Vaccine Company.

Introduction Familial hypocalciuric hypercalcemia is a rare autosomal dominant disorder characterized by asymptomatic and non-progressive hypercalcemia due to mutations of the calcium-sensing receptor gene. Disorders of calcium metabolism are very common in the elderly, and they can coexist with familial hypocalciuric hypercalcemia in affected families. Case presentation We describe an Irish family with hypercalcemia and hypocalciuria. The proband, an 80-year-old Irish woman, presented with hypercalcemia, relative hypocalciuria, and an elevated parathormone level. She also had chronic kidney disease stage 3 and vitamin D deficiency. Two of her sons were also found to be hypercalcemic and hypocalciuric. DNA sequencing identified a novel missense inactivating mutation in the calcium sensing-receptor gene of the proband and her two hypercalcemic sons. Conclusion Familial hypocalciuric hypercalcemia due to a novel mutation in the calcium-sensing receptor gene was diagnosed in the proband and her two sons. Disorders of calcium metabolism can be multifarious in the elderly. We suggest that testing first degree relatives for calcium levels and DNA sequencing may have a role in the assessment of elderly patients with parathormone-related hypercalcemia.

Over the past several years, many tumor markers, including cell surface antigens, T-antigen, ras p55, and ras p52 proteins, have been studied as potential tumor markers of bladder cancer. The lack of specificity and inconsistency of these markers led us to develop a new method for studying the urinary excretion of autocrine motility factor (uAMF) and tumor cell collagenase stimulating factor (TCSF) in 24-hour and first morning voided specimens. AMF is a glycoprotein secreted by the malignant cells and is responsible for cell locomotion, a key event in invasion and metastases of the malignant cells. TCSF is a membrane bound glycoprotein of tumor cells that stimulates fibroblast collagenase production. We have utilized an enzyme-linked immunoabsorption assay to detect the levels of uAMF and TCSF in urine samples collected from normal volunteers, patients with benign diseases, and patients with bladder cancer. Our data indicate that urinary concentrations of uAMF and TCSF are elevated in patients with bladder cancer. Furthermore, the levels of uAMF and TCSF are more elevated in invasive tumors as compared with benign counterparts. We have localized uAMF and TCSF in bladder cancer cells, utilizing immunohistologic techniques.