Contribution of real-time PCR to Plasmodium species identification and to clinical decisions: a nationwide study in a non-endemic setting.

Treatment choice for patients with malaria in Israeli hospitals is based on microscopy and rapid diagnostic tests (RDTs). Here, we demonstrate the cumulative value of real-time polymerase chain reaction (PCR) in optimizing the treatment of malaria. Between January 2009 and December 2015, 451 samples from 357 patients were tested in our laboratory using a real-time PCR assay. Hospital laboratory results (without real-time PCR) were compared to those obtained in our laboratory. A total of 307 patients had a malaria-positive laboratory finding in the hospital. Out of those, 288 were confirmed positive and 19 negative using real-time PCR. Two negative hospital results were found to be positive by real-time PCR. More specifically, of 153 cases positive for Plasmodium falciparum by real-time PCR, only 138 (90%) had been correctly identified at the hospitals. Similarly, 66 (67%) of 99 cases positive for P. vivax, 2 (11%) of 18 cases positive for P. ovale, and 3 (30%) of 10 cases positive for P. malariae had been correctly identified. Of 10 cases of mixed infection, only one had been identified as such at the hospital. Thus, real-time PCR was required for correct identification in 81 (28%) out of 290 positive cases. In 52 (18%) of those, there was an erroneous categorization of relapsing versus non-relapsing parasites. In a nationwide study, we found that the use of real-time PCR is definitely beneficial and may change the decision regarding the choice of treatment.

Antibacterial activity of eravacycline (TP-434), a novel fluorocycline, against hospital and community pathogens.

Eravacycline (TP-434 or 7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline) is a novel fluorocycline that was evaluated for antimicrobial activity against panels of recently isolated aerobic and anaerobic Gram-negative and Gram-positive bacteria. Eravacycline showed potent broad-spectrum activity against 90% of the isolates (MIC90) in each panel at concentrations ranging from ≤0.008 to 2 µg/ml for all species panels except those of Pseudomonas aeruginosa and Burkholderia cenocepacia (MIC90 values of 32 µg/ml for both organisms). The antibacterial activity of eravacycline was minimally affected by expression of tetracycline-specific efflux and ribosomal protection mechanisms in clinical isolates. Furthermore, eravacycline was active against multidrug-resistant bacteria, including those expressing extended-spectrum ß-lactamases and mechanisms conferring resistance to other classes of antibiotics, including carbapenem resistance. Eravacycline has the potential to be a promising new intravenous (i.v.)/oral antibiotic for the empirical treatment of complicated hospital/health care infections and moderate-to-severe community-acquired infections.

Structures of human dihydroorotate dehydrogenase in complex with antiproliferative agents.

BACKGROUND: Dihydroorotate dehydrogenase (DHODH) catalyzes the fourth committed step in the de novo biosynthesis of pyrimidines. As rapidly proliferating human T cells have an exceptional requirement for de novo pyrimidine biosynthesis, small molecule DHODH inhibitors constitute an attractive therapeutic approach to autoimmune diseases, immunosuppression, and cancer. Neither the structure of human DHODH nor any member of its family was known. RESULTS: The high-resolution crystal structures of human DHODH in complex with two different inhibitors have been solved. The initial set of phases was obtained using multiwavelength anomalous diffraction phasing with selenomethionine-containing DHODH. The structures have been refined to crystallographic R factors of 16.8% and 16.2% at resolutions of 1. 6 A and 1.8 A for inhibitors related to brequinar and leflunomide, respectively. CONCLUSIONS: Human DHODH has two domains: an alpha/beta-barrel domain containing the active site and an alpha-helical domain that forms the opening of a tunnel leading to the active site. Both inhibitors share a common binding site in this tunnel, and differences in the binding region govern drug sensitivity or resistance. The active site of human DHODH is generally similar to that of the previously reported bacterial active site. The greatest differences are that the catalytic base removing the proton from dihydroorotate is a serine rather than a cysteine, and that packing of the flavin mononucleotide in its binding site is tighter.

Different combinations of genetic/epigenetic alterations inactivate the p53 and pRb pathways in invasive human bladder cancers.

Inactivation of both the pRb (pRb-cyclin D1/cyclin-dependent kinase 4/6-p16) and p53 (p53-p21(WAF1)-p14(ARF)) pathways is thought to be essential for immortalization in vitro and malignant transformation in vivo. We identified different combinations of pRb and p53 pathway alterations in 12 invasive transitional cell carcinomas (TCCs) and addressed the functional significance of the different combinations observed. Results showed four combinations of alterations including -pRb/-p53 (ie., pRb inactivated in the pRb pathway and p53 inactivated in the p53 pathway; four TCCs), -p16/-p53 (four TCCs), -p16/-p21(WAF1) (one TCC), and -p16/ -p14(ARF) (two TCCs). These groups include two new combinations (ie., -p16/-p53 and -p16/-p21(WAF1)) not reported previously for TCCs. An alteration in the key components of the p53 pathway was not detected in one invasive TCC that had inactivated p16. Note that all four TCCs with inactivated pRb had mutant p53; thus, the combinations of -pRb/ -p21(WAF1) and -pRb/-p14(ARF) were not observed. Only two of eight TCCs with altered p16 had concomitant p14(ARF) loss, demonstrating that simultaneous inactivation of these two 9p21INK4a tumor suppressor genes is not obligatory. To determine the biological phenotypes of TCCs with different combinations of pRb and p53 pathway alterations, their downstream responses to gamma radiation were studied in vitro. As expected, none of eight TCCs with mutant p53 responded to gamma radiation by elevation of p53, p21(WAF1), or mdm2 or by cell cycle arrest. Only two of four TCCs with wild-type p53 and wild-type pRb (the combination of -p16/-p14(ARF)) showed normal downstream responses to gamma radiation and underwent cell cycle arrest. Two TCCs with wild-type pRb and wild-type p53 (the combination of -pl6/-p21(WAF1) and one TCC with -p16) failed to show cell cycle arrest in response to radiation. This was attributed to the absence of p21(WAF1) in one TCC. In summary, these data support a model of invasive bladder cancer pathogenesis in which both the pRb and p53 pathways are usually inactivated and the biology of the tumor is impacted by the mechanism of their inactivations.

Sequence and genetic organization of a Bacillus subtilis operon encoding 2,3-dihydroxybenzoate biosynthetic enzymes.

Under iron-limiting conditions, Bacillus subtilis (Bs) produces the siderophore 2,3-dihydroxybenzoate (DHB) to acquire extracellular iron. In Escherichia coli (Ec), DHB is a precursor of the siderophore enterobactin, which suggested that Bs may possess similar biosynthetic enzymes. The sequences of two overlapping Bs clones capable of complementing Ec enterobactin mutants [Grossman, T.H., Tuckman, M., Ellestad, S. and Osburne, M.S. (1993) Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: Relationship between B. subtilis sfpo and Escherichia coli entD genes. J. Bacteriol. 175, 6203-6211] were analyzed and five open reading frames were identified. These genes are located near 291 degrees on the Bs chromosome and have been termed dhbA, dhbC, dhbE, dhbB and dhbF, based on similarities to Ec ent homologs. Amino-acid identities between gene product homologs are: EntA and DhbA, 41%; EntC and DhbC, 35%; EntE and DhbE, 48%; EntB and DhbB, 54%; and EntF and DhbF, 29%. DhbC is also 35% identical to the Bs menaquinone-specific isochorismate synthase, MenF, illustrating an example of gene duplication. Operon disruption studies suggested that the dhb genes comprise an operon of at least four genes.

Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability.

E. coli recombinant expression systems that utilize lac operon control elements to modulate gene expression are known to produce some amount of uninduced (leaky) gene expression. Previously, we showed that high levels of uninduced gene expression was a major cause of instability in the pET expression system. We show here that the pET system, in which the phage T7 RNA polymerase gene is expressed via lac operon control elements, exhibits leaky expression that increases markedly as cells grown in complex medium enter stationary phase. Moreover, we found that this phenomenon occurs with the chromosomal lac operon as well. Further investigation revealed that stationary phase leaky expression requires cyclic AMP, and that substantial leaky expression could be effected in log phase cells by adding cyclic AMP and acetate at pH6.0. Finally, a comparison of otherwise isogenic cya and wild-type hosts showed that expression stability and plasmid maintenance in the cya host is greatly enhanced, even when cells are passaged repeatedly in non-selection medium. These findings both provide a method to enhance the stability of lac-based recombinant expression systems, and suggest that derepression of the lac operon in the absence of inducer may be part of a general cellular response to nutrient limitation.

High-level production of a secreted, heterodimeric alpha beta murine T-cell receptor in Escherichia coli.

For structural studies, high-level production of properly folded, disulfide-linked, unglycosylated protein in E. coli is an attractive alternative to production in eukaryotic systems. We describe here the production of heterodimeric, murine D10 T-cell receptor (sD10TCR) in E. coli as a secreted leucine zipper (LZ) fusion protein. Two genes, one (alpha-acid) encoding the alpha-chain variable and constant domains (V alpha and C alpha) of D10 TCR fused to an LZ 'acid' encoding sequence and the other (beta-base) encoding the beta-chain variable and constant domains (V beta and C beta) fused to an LZ 'base' encoding sequence, were co-expressed from a bacteriophage T7 promoter as a dicistronic message. Secreted alpha-acid and beta-base proteins formed proper inter- and intra-chain disulfide bonds in the periplasm, bypassing the need for in vitro protein refolding. Complementary LZ sequences facilitated the formation of alpha beta heterodimers. sD10TCR-LZ was purified by affinity chromotography using a D10 TCR clonotype-specific monoclonal antibody (mAb 3D3). Typical yields of purified protein were 4-5 mg/l of culture. Purified sD10TCR-LZ was reactive with a panel of conformationally sensitive TCR-specific monoclonal antibodies, consistent with its conformational integrity and appeared to be suitable for structural studies by X-ray crystallography or NMR spectroscopy.

Production of secreted, soluble human two-domain CD4 protein in Escherichia coli.

The two-domain form of recombinant soluble human CD4 (rsCD4(183)) has been used for structural studies and to probe the interaction of CD4 with its ligands. rsCD4(183) has generally been produced in Escherichia coli in the form of inclusion bodies. The generation of conformationally native protein from these inclusion bodies is a time-consuming and inefficient process, requiring a refolding step. Here, we describe a procedure for producing 2-4 mg of secreted, conformationally native rsCD4(183) per liter of E. coli, completely bypassing the requirement for protein refolding in vitro. Furthermore, the yield of active protein is comparable to that reported for expression systems that generate inclusion bodies.

A randomized study of adjuvant chemotherapy for cancer of the upper aerodigestive tract.

A prospective, randomized trial of induction chemotherapy in advanced squamous cell carcinomas of the upper aerodigestive tract (UAD) was conducted between July 1979 and September 1982. Eighty-three patients with locally advanced Stage III-IV tumors received standard treatment (STD RX; defined as preoperative irradiation and radical excision or irradiation alone), or induction chemotherapy (CTX) followed by STD RX. Chemotherapy consisted of two cycles of bleomycin (30 units/day by continuous infusions Days 1-4), cyclophosphamide (200 mg/m2 IV Days 1-5), methotrexate (30 mg/m2 Days 1 + 5), and 5-fluorouracil (400 mg/m2 IV Days 1-5). Response to CTX was complete in 2 and partial (greater than 50% reduction) in 27; the overall response rate was 68%. Tumor clearance was documented in 30/40 STD RX patients at completion of irradiation and/or surgery and in 24/43 CTX patients (17/29 responders, 7/14 non-responders). Freedom from local-regional disease was noted at 2 years in 53% STD RX and 35% CTX patients (p less than .06). CTX patients had a higher proportion of local-regional persistence and recurrence. The difference was apparent only in the subset of patients treated with primary irradiation; local-regional control following irradiation and surgery was equal in STD RX and CTX groups. Survival at 2 years was 43% STD RX and 31% CTX. Disease-free survival in those with clearance was 64% STD RX and 59% CTX. Induction chemotherapy did not improve tumor clearance or survival in this series. Caution regarding local-regional control with CTX and primary irradiation is noted.

Simpson-Golabi-Behmel syndrome: genotype/phenotype analysis of 18 affected males from 7 unrelated families.

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked overgrowth disorder recently shown to be caused by mutations in the heparan sulfate proteoglycan GPC3 [Pilia et al., Nat Genet; 12:241-247 1996]. We have used Southern blot analysis and polymerase chain reaction amplification of intra-exonic sequences to identify four new GPC3 mutations and further characterize three previously reported SGBS mutations. De novo GPC3 mutations were identified in 2 families. In general, the mutations were unique deletions ranging from less than 0.1 kb to more than 300 kb in length with no evidence of a mutational hot spot discerned. The lack of correlation between the phenotype of 18 affected males from these 7 families and the location and size of the GPC3 gene mutations suggest that SGBS is caused by a nonfunctional GPC3 protein.

Hearing loss in former prisoners of war of the Japanese.

OBJECTIVE: To describe the prevalence, degree, and types of hearing loss present in a group of older American veterans who had been prisoners of war of the Japanese. DESIGN: A descriptive study. SETTING: A Veterans Affairs university hospital. PARTICIPANTS: Seventy-five male veterans, mean age 68 (+/- 3.6) years. INTERVENTIONS: Hearing aids were prescribed for eight veterans. MEASUREMENTS: Subjects were examined, and pure tone air and bone conduction, speech reception threshold, and speech discrimination were determined. Results were compared with age- and sex-matched controls from the largest recent American population study of hearing loss. RESULTS: 95% of subjects had been imprisoned longer than 33 months. Starvation conditions (100%), head trauma (85%), and trauma-related loss of consciousness (23%) were commonly reported. A total of 73% complained of hearing loss, and 29% (22/75) dated its onset to captivity. Most of those with the worst losses in hearing and speech discrimination were found in this subgroup. When the entire group was compared with published age- and sex-matched controls from the Framingham Study, no significant differences were found. CONCLUSIONS: We advocate screening examinations and long-term follow-up of populations with similar histories of starvation, head trauma, and torture.

The incidence and diagnosis of secondary esophageal carcinoma in the head and neck cancer patient.

The historical development of additional primary neoplasms of the esophagus is traced. To determine the incidence and time of appearance of these esophageal malignancies, a retrospective study of 790 head and neck cancer patients was conducted. In addition, a prospective study of 268 head and neck cancer patients was undertaken to determine the optimum diagnostic approach for esophageal neoplasms. All patients in the prospective study had a pharyngoesophagogram plus esophagoscopy as part of the initial diagnostic evaluation. The false negative rate of the pharyngoesophagogram was 72.2% in patients with esophageal pathology. In patients with esophageal malignancies it was 80%. Esophagoscopy should be a part of the initial evaluation of every patient with an upper aerodigestive tract primary malignancy.

The diagnostic accuracy of pharyngoesophagram compared to esophagoscopy in patients with head and neck cancer.

Two hundred and fifty-four patients with head and neck cancer were entered into a prospective study comparing the results of pharyngoesophagram to esophagoscopy. All patients had pharyngoesophagram deemed adequate to evaluate the esophagus and rigid esophagoscopy to at least 30 cm from the upper incisor teeth. In 239 (94.1%) patients both the pharyngoesophagram and esophagoscopy were negative. Four esophageal tumors were found. Only one of these tumors (25%) was detected on pharyngoesophagram. We believe that the pharyngoesophagram should be a part of the initial work-up of every head and neck cancer patient and should precede esophagoscopy. However, the barium swallow cannot be relied upon exclusively to detect small simultaneous second primary esophageal malignancies.

Rhinitis medicamentosa.

The diagnosis of rhinitis medicamentosa was made in 130 patients seen over a 10 year period from July 1967 to June 1977. There was an incidence of 1% in our otolaryngological practice. Patients had been taking the causal medication for an average of 21.4 months. There were 73 males and 57 females with the peak incidence in young and middle-age adults. The primary offending medications were decongestant nasal sprays in 85 patients, decongestant drops in 33, and a combination of these drugs in 12 patients. The major reasons for self-medication were 1. deviated nasal septum in 40 patients, 2. an acute upper respiratory infection in 33, 3. allergy in 18, 4. miscellaneous causes in 24 and 5. unknown in 15 patients. The initial management in addition to avoidance of the medication consisted of systemic antibiotics, decongestants, antihistamines, and sedatives depending on the severity of the rhinitis and the presence of secondary infection. Later treatment consisted of correction of the deviated septums, allergic management, and supportive care. Eight patients were considered to have complications of the disease by development of chronic ethmoiditis and nasal polyposis. The pharmacologic properties of the causal agents are thoroughly reviewed as they relate to the pathogenesis of this disease. It is felt that the ready commercial availability and limited clinical value of the topical nasal sprays and drops represents a certain risk to all patients using them.

Adenoid cystic carcinoma of the submaxillary gland.

A review of submaxillary gland neoplasms over a 21 year period revealed 15 malignancies and 7 benign tumors. All of the malignancies occurred in females, and 11 of these were adenoid cystic carcinoma. Two of the 11 were found to have infiltrating ductal carcinoma of the breast (1 pre and 1 postdiagnosis of the submaxillary carcinoma) and 3 had benign breast disease. While previous reports have suggested an association of parotid gland neoplasia and breast cancer, this is the first known report of an association between adenoid cystic carcinoma of the submaxillary gland and cancer of the breast. The discussion of adenoid cystic carcinoma of the submaxillary gland emphasizes the increased frequency of this disease in females, its association with breast disease, and also experimental submaxillary gland neoplasia.

Cisplatin and fluorouracil as neoadjuvant therapy in head and neck cancer. A preliminary report.

A randomized, prospective trial utilizing cisplatin and fluorouracil as neoadjuvant chemotherapy in the treatment of advanced squamous cell carcinomas of the upper aerodigestive tract was initiated in January 1983. Sixty patients were stratified by site (oral cavity, 19; larynx, 14; hypopharynx, 14; oropharynx, 11; nasopharynx, one; and paranasal sinuses, one) and by stage (III, 19; IV, 41), and then randomized to receive either standard treatment (defined as preoperative irradiation followed by radical excision or irradiation alone) or adjuvant chemotherapy followed by standard treatment. An additional three patients were entered into the study, but withdrew. Chemotherapy consisted of three cycles for those patients in whom an objective tumor response was observed; nonresponders received standard treatment. Response to chemotherapy was complete in five and partial (greater than 50%) in 18 patients, for an overall response rate of 85%. The follow-up for surviving patients was a minimum of 24 months and a maximum of 44 months. Survival was compared for patients in both treatment groups according to the method of Lee and Desu. Despite excellent tumor response, actuarial survival was 70% in the standard treatment group as opposed to 56% in the experimental group. It was therefore evident that the high response rates reported in previous pilot studies do not necessarily result in improved survival in these cancers.

Randomized study of adjuvant chemotherapy for head and neck cancer.

The ability of surgery and radiotherapy to control advanced squamous cell carcinoma of the head and neck has reached its maximal potential. We initiated a randomized, prospective, stratified study of adjuvant chemotherapy. Patients with stage II disease of the pyriform sinus and stage II and IV disease of the oral cavity, larynx, hypopharynx, oropharynx, nasopharynx, and paranasal sinuses were eligible. Patients were randomized to receive either standard therapy alone or two courses of 5-fluorouracil (B-CMF) chemotherapy prior to and two courses after the completion of standard therapy. Standard therapy consisted of preoperative irradiation followed by radical surgery. Of 133 patients with advanced disease, 83 were included in the study--43 in the chemotherapy group and 40 in the control group. Rates of residual and recurrent disease, as well as distant metastases, were similar for the two groups. The survival rates of patients without persistent disease at the end of treatment showed no significant difference for the two groups. The study has been discontinued because statistical analysis indicated that the addition of more patients would not materially increase the statistical significance of the study.

Delayed aerodigestive tract complications following combined therapy for thyroid cancer.

Radical surgery for papillary adenocarcinoma of the thyroid has been associated with a significant incidence of complications. In some instances, postoperative irradiation is given when there is some suspicion of persistent or occult disease, although thyroid suppression and ablative radioiodine therapy have proved to be very effective adjuvants to surgery. Three patients with papillary adenocarcinoma of the thyroid developed severe, delayed complications 25, 7, and 2 years, respectively, after treatment with primary radical surgery and postoperative irradiation. The degree of injury to the aerodigestive tract as a result of the surgery and irradiation therapy makes treatment difficult regardless of the modality. The possible mechanisms that cause these complications, along with proposed methods of treatment, are discussed.

Association of laryngeal and pulmonary malignancies: a continuing challenge.

Over a 10-year period, 790 patients with squamous cell carcinoma of the head and neck were treated at The Medical College of Wisconsin Affiliated Hospitals and were followed for a minimum of 7 years. Of the 218 patients with index primary laryngeal tumors, 43 (19.7%) developed additional malignancies in the head and neck, esophagus, or lung. Secondary lung tumors were the most common, occurring in 23 patients (10.6%). Of the 218 patients with index primary laryngeal carcinoma, 113 were treated successfully and never developed a recurrence of the original tumor. Twenty-one second primary lung malignancies developed in this group of successfully treated laryngeal tumor patients. The occurrence of these pulmonary malignancies was distributed fairly evenly over time. Three patients developed second primary lung tumors more than 7 years after initial treatment. The appearance of a secondary malignancy in the lung had a devastating effect upon survival. None of our patients survived more than 2 years after detection of the lung lesion. The relatively high incidence and delayed onset of second primary lung tumors in this group call into question the concept of 5-year "cures." Our challenge for the future should be the prevention and early detection of these second primary lung tumors.

Complications associated with a narrow bore nasogastric tube.

The use of a new narrow bore nasogastric feeding tube with stylet has resulted in two cases of misplacement with traumatic laceration of the visceral pleura. Diagnoses were made by chest x-ray film, one case immediately and the other 24 hours later. In both cases, the narrow bore tube was inserted asymptomatically by a graduate physician. Detailed case reports of both patients are presented, those patients at risk for abnormal placement of this nasogastric tube are discussed, and new guidelines for safer use are proposed.