Stage-specific expression of the proline-alanine transporter in the human pathogen Leishmania.

Leishmania are obligatory intracellular parasites that cycle between the sand fly midgut (extracellular promastigotes) and mammalian macrophage phagolysosomes (intracellular amastigotes). They have developed mechanisms of adaptation to the distinct environments of host and vector that favor utilization of both proline and alanine. LdAAP24 is the L. donovani proline-alanine transporter. It is a member of Leishmania system A that translocates neutral amino acids. Since system A is promastigote-specific, we aimed to assess whether LdAAP24 is also expressed exclusively in promastigotes. Herein, we established that upon exposing L. donovani promastigotes to amastigote differentiation signal (pH 5.5 and 37 °C), parasites rapidly and completely degrade LdAAP24 protein in both axenic and in spleen-derived amastigotes. In contrast, LdAAP24 mRNA remained unchanged throughout differentiation. Addition of either MG132 or Bafilomycin A1 partially inhibited LdAAP24 protein degradation, indicating a role for both lysosome- and proteasome-mediated degradation. This work provides the first evidence for post-translational regulation of stage-specific expression of LdAAP24.

Effectiveness of rotavirus pentavalent vaccine under a universal immunization programme in Israel, 2011-2015: a case-control study.

OBJECTIVES: The use of rotavirus pentavalent vaccine (RotaTeq®) as a sole vaccine within rotavirus universal immunization programmes remains limited. We examined the effectiveness of RotaTeq in preventing rotavirus gastroenteritis (RVGE) hospitalization in Israel, after the introduction of universal immunization against the disease. METHODS: A test-negative case-control study included age-eligible children for universal RotaTeq immunization (aged 2-59 months, born in 2011-2015). Cases (n = 98) were patients who tested positive for rotavirus by immunochromatography; those who tested negative (n = 628) comprised the control group. Information on rotavirus immunization history was obtained through linkage with a national immunization registry. Vaccination status was compared between cases and controls, adjusted odds ratios (aORs) were obtained from logistic regression models, and vaccine effectiveness calculated as (1 - aOR)*100. RESULTS: Immunization with RotaTeq was less frequent in RVGE cases (73.5%) than in controls (90.1%), p < 0.001; this association persisted after controlling for potential confounders. Effectiveness of the complete vaccine series was estimated at 77% (95% confidence interval (CI): 49-90) in children aged 6-59 months, and 86% (95% CI: 65-94) in children aged 6-23 months; whereas for the incomplete series, the respective estimates were 72% (95% CI: 28-89) and 75% (95% CI: 30-91). Vaccine effectiveness was estimated at 79% (95% CI: 45-92) against G1P[8]-associated RVGE hospitalizations and 69% (95% CI: 11-89) against other genotype-RVGE hospitalizations. CONCLUSIONS: High effectiveness of RotaTeq as the sole rotavirus vaccine in a universal immunization programme was demonstrated in a high-income country. Although partial vaccination conferred protection, completing the vaccine series is warranted to maximize the benefit.

Dynamics of childhood invasive meningococcal disease in Israel during a 22-year period (1989-2010).

AIM: To describe the dynamics in the incidence of childhood invasive meningococcal disease (IMD) in Israel during a 22-year period (1989-2010). METHODS: A longitudinal prospective surveillance in all 27 medical centers with pediatric services in Israel. All cases of children <15 years old with positive blood/cerebrospinal fluid (CSF) culture for Neisseria meningitidis were reported. Demographic, clinical, and bacteriological data were recorded. Meningococcal vaccine was not routinely given to Israeli children during the study period. RESULTS: The mean age ± standard deviation (SD) among the 743 cases was 40.7 ± 40.2 months. The mean yearly incidence/100,000 was 2.0 ± 0.8. Age-specific incidences were 8.7 ± 2.8, 2.9 ± 1.5, and 0.8 ± 0.5 for children <1, 1-4, and >4 years old, respectively. The overall incidence decreased significantly from 3.7 in 1989 to 1.5 in 2010. Meningitis constituted 69.2 % of all cases. The most common serogroups were: B (76.9 %), C (10.9 %), Y (8.0 %), and W(135) (2.9 %). 78.6 % of all serogroup B isolates were from children <5 years old (p < 0.01). Serogroup C was found mainly in children ≥5 years old (63.4 %). The case fatality rates (CFRs) for children <1, 1-4, >4 years old, and the total study population were 9.2, 12.3, 7.7, and 9.9 %, respectively. CFRs were higher for children without meningitis (14.9 %) compared to children with meningitis (7.9 %) (p < 0.01). CONCLUSIONS: Overall, and for serogroups B and W135, childhood IMD rates decreased significantly in Israel during the study period, without routine vaccine usage. The most common serogroup in all age groups was B, which was most prevalent in children <5 years old. No change in the trend of the overall CFR was noted during the study period.

Liposomal amphotericin B treatment of cutaneous leishmaniasis due to Leishmania tropica.

BACKGROUND: Cutaneous leishmaniasis (CL) is endemic in Israel, and in the past, has been attributed almost exclusively to Leishmania major. Over the last decade or so, an increase in Leishmania tropica (L. tropica) infections has occurred in several regions of Israel. Topical treatment of Old World CL is usually the rule, however, in some cases systemic treatment is indicated. Liposomal amphotericin B (L-AmB) is efficacious and safe for treating visceral leishmaniasis but its role in treating various forms of CL is yet to be defined. In this study, we summarize the efficacy and safety of L-AmB treatment in a series of Israeli patients with L. tropica infection. METHODS: Cases of PCR-proven CL caused by L. tropica were treated in an outpatient setting. Treatment schedule consisted of five consecutive days of 3 mg/kg L-AmB, followed by a sixth dose on day 10. RESULTS: Thirteen consecutive patients (11 men, two women), received L-AmB. Mean age was 15.3 years; of the 13 patients, 85% had facial lesions. Six had previously failed intralesional sodium stibogluconate treatment and four had failed topical paromomycin treatment. Eleven of 13 patients (84%) achieved complete clinical cure within 2 months. Mean follow-up of 11 months revealed no relapses. Side effects were mild and none terminated treatment prematurely. LIMITATIONS: A non-randomized study, with a small number of patients. CONCLUSION: Liposomal amphotericin B treatment for L. tropica is effective, well tolerated and cost beneficial in countries where cost of hospital-care is significant.

Nontuberculous mycobacteria in cystic fibrosis associated with allergic bronchopulmonary aspergillosis and steroid therapy.

Nontuberculous mycobacterial (NTM) infection, particularly due to Mycobacterium abscessus, is an emerging disease that can be relentlessly progressive, particularly in cystic fibrosis (CF) patients. The risk factors that were associated with this increasingly symptomatic infection in a group of CF patients were investigated. A total of 139 CF patients aged 2-52 yrs were reviewed. Sputum was cultured for NTM annually or whenever clinical deterioration was unexplained. In total, 12 patients (8.6%) had positive cultures and six (4.3%) met the criteria for NTM pulmonary disease (five with M. abscessus). Five had allergic bronchopulmonary aspergillosis (ABPA) compared with one out of 133 patients without NTM disease. Five had received systemic steroids (four as a treatment for ABPA) compared with only one out of 133 without NTM lung disease. All six NTM patients deteriorated markedly following mycobacterial infection, and forced expiratory volume in one second dropped 18-46%. Despite prolonged triple antibiotic therapy, M. abscessus was not eradicated, and four out of six did not return to baseline clinically. In conclusion, severe nontuberculous mycobacterial lung disease, particularly with Mycobacterium abscessus, is becoming a perplexing challenge in cystic fibrosis patients. Allergic bronchopulmonary aspergillosis and systemic steroids appear to be risk factors, although small patient numbers limit this to a descriptive observation. When pulmonary condition deteriorates, increased surveillance for mycobacteria would enable prompt diagnosis and treatment.

Enzyme immunoassay for the diagnosis of cat-scratch disease defined by polymerase chain reaction.

Whole-cell immunofluorescent antibody (IFA) tests for detection of anti-Bartonella henselae immunoglobulin (Ig) G are commonly used to diagnose cat-scratch disease (CSD). The need to cultivate B. henselae in Vero cells for antigen preparation and the absence of routinely applied IFA assays for IgM constitute the major disadvantages of this form of test. We describe the results of an enzyme immunoassay (EIA) for IgM and IgG that used N-lauroyl-sarcosine-insoluble outer membrane antigens from agar-grown B. henselae performed in 84 patients with definite CSD (regional lymphadenitis, cat contact, and > or =1 confirmatory test: polymerase chain reaction, skin test, or B. henselae culture). Although this method has been used as a diagnostic tool in several case reports, it has not previously been evaluated in a large study of definitively proven CSD cases. Results of this study indicate that the EIA described herein can play an important role in the serodiagnosis of CSD, although improvement of the sensitivity, particularly that of the IgM, would be desirable.

DNA amplification for the diagnosis of cat-scratch disease in small-quantity clinical specimens.

Diagnosis of cat-scratch disease (CSD) by polymerase chain reaction (PCR) of lymph node fineneedle aspiration (FNA) and primary lesion specimens can be difficult owing to the minute amount of available material. A PCR assay specifically suited to test these specimens was developed. First, small-quantity (10 microL) samples were prepared from 17 CSD-positive and 16 CSD-negative specimens, and DNA extraction and amplification from these samples were compared using 3 methods. Sensitivity and specificity of PCR were 100% using material collected on glass microscope slides and by using Qiagen (Hilden, Germany) columns for DNA extraction. Then, this method was used to test 11 archival glass microscope slides of FNA (7 malignant neoplasms, 4 undiagnosed lymphadenitis) and 2 primary lesion specimens. Two of the 4 lymphadenitis samples and the 2 primary lesion specimens were PCR positive. The technique presented could facilitate CSD diagnosis from a wider range of clinical samples.

Novel Intracellular SbV reducing activity correlates with antimony susceptibility in Leishmania donovani.

The standard treatment of human visceral leishmaniasis involves the use of pentavalent antimony (Sb(V)). Its mechanism of action is unknown because of the limited information available about intracellular antimony metabolism and about the genes that regulate these processes. Herein, flow injection-inductively coupled plasma mass spectrometry (ICP-MS), flow injection hydride generation ICP-MS, and ion chromatography ICP-MS were used to measure antimony accumulation and intracellular metabolism in the human protozoan parasite Leishmania donovani. Amastigotes (the intracellular form) and promastigotes (the extracellular form) accumulate Sb(V) and Sb(III) via separate transport systems. Stage-specific intracellular Sb(V) reducing activity was apparent in amastigotes, which reduced the negligibly toxic Sb(V) to highly toxic Sb(III). This amastigote-specific reducing activity was deficient in the Pentostam-resistant mutant L. donovani Ld1S.20. These data indicate that parasite susceptibility to Sb(V) correlates with its level of Sb(V) reducing activity. Also, in promastigotes of both wild-type L. donovani and the Pentostam-resistant mutant L. donovani Ld1S.20, Sb(V) inhibited the toxicity of Sb(III) but not of As(III). Both Sb(V) and Sb(III) were toxic to wild-type amastigotes. However, as observed in promastigotes, in mutant amastigotes Sb(V) inhibits Sb(III) but not As(III) activity. Anion exchange chromatography showed that intracellular antimony metabolism occurred in both promastigotes and amastigotes. These data demonstrate that the interaction between the two antimony oxidation states occurs intracellularly, within the parasite. The results also indicate that Sb(V) anti-leishmanial activity is dependent on its reduction to Sb(III). The mechanism of this novel intracellular Sb(V) reduction has yet to be identified, and it may or may not be enzymatic. This is the first description of intracellular Sb(V) reducing activity in Leishmania as well as in any prokaryotic or eukaryotic cell.

Safety and immunogenicity of two different lots of the oral, killed enterotoxigenic escherichia coli-cholera toxin B subunit vaccine in Israeli young adults.

Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of diarrhea among Israeli soldiers serving in field units. Two double-blind placebo-controlled, randomized trials were performed among 155 healthy volunteers to evaluate the safety and immunogenicity of different lots of the oral, killed ETEC vaccine consisting of two doses of whole cells plus recombinantly produced cholera toxin B subunit (rCTB). The two doses of vaccine lot E005 and the first dose of vaccine lot E003 were well tolerated by the volunteers. However, 5 (17%) vaccinees reported an episode of vomiting a few hours after the second dose of lot E003; none of the placebo recipients reported similar symptoms. Both lots of vaccine stimulated a rate of significant antibody-secreting cell (ASC) response to CTB and to colonization factor antigen I (CFA/I) after one or two doses, ranging from 85 to 100% and from 81 to 100%, respectively. The rate of ASC response to CS2, CS4, and CS5 was slightly lower than the rate of ASC response induced to CTB, CFA/I, and CS1. The second vaccine dose enhanced the response to CTB but did not increase the frequencies or magnitude of ASC responses to the other antigens. The two lots of the ETEC vaccine induced similar rates of serum antibody responses to CTB and CFA/I which were less frequent than the ASC responses to the same antigens. Based on these safety and immunogenicity data, an efficacy study of the ETEC vaccine is under way in the Israel Defense Force.

Measles epidemic in Israel-successful containment in the military.

BACKGROUND: Measles vaccination at ages 12-15 months is a routine part of standard health care in developed countries. Nonetheless, the prevention and control of measles outbreaks remain a challenge, owing to incomplete or variable compliance with immunization programs and primary vaccine failure (approximately 5%). In Israel, vaccination coverage against measles is high, yet sero-epidemiological studies conducted in the early 1990s showed that 15% of 18-year-olds were unprotected. METHODS: 1994 there was a countrywide epidemic of measles, which spread to the military. The Israel Defense Forces Medical Corps immediately launched a wide-scale vaccination campaign, targeting primarily field units and training bases, where crowded living conditions are the rule. RESULTS: The immunization campaign led to an abrupt cessation of morbidity in the military. In the civilian sector, where no intervention was undertaken, the epidemic continued for another 4 months. CONCLUSIONS: Institutional measles outbreaks, especially in the presence of crowded conditions or high contact rates, may be effectively controlled by mass vaccination.

Unusual eruption as a presenting symptom of cat scratch disease.

Cat scratch disease (CSD) is a common infectious cause of subacute regional lymphadenopathy. Bartonella henselae is the principal etiologic agent. About 10% of CSD patients experience atypical manifestations, including rashes. The most common cutaneous manifestation of CSD is a papule at the inoculation site. We report a case of CSD presenting with an eruption on the upper trunk, reminiscent of Sweet's syndrome, accompanied by lymphadenopathy, arthralgia, and fever. Response to systemic corticosteroids was remarkable. Histopathologic findings refuted the diagnosis of Sweet's syndrome. Identification of anti-B henselae antibodies and B henselae DNA in the affected lymph node confirmed the diagnosis of CSD. This is a first report of extensive papuloedematous eruption as a cutaneous manifestation of CSD. Accurate diagnosis is possible due to the availability of serological tests and DNA amplification techniques.

Stage-specific activity of pentavalent antimony against Leishmania donovani axenic amastigotes.

The standard treatment of human visceral leishmaniasis involves the use of pentavalent antimony (SbV) compounds. In recent years increasing numbers of clinical failures of treatment with SbV have been reported, probably due to the development of parasite resistance to this compound. The mode of action and mechanisms of resistance to SbV have not been fully elucidated. In the present study an axenic amastigote culture was used to study the in vitro responses of Leishmania donovani to SbV. Susceptibility to both sodium stibogluconate and meglumine antimoniate was found to be stage specific. Amastigotes were 73 to 271 times more susceptible to SbV than were promastigotes. As opposed to SbV, trivalent antimony (SbIII) was similarly toxic to both developmental stages. When promastigotes were transformed to amastigotes, susceptibility to meglumine antimoniate developed after 4 to 5 days, upon the completion of differentiation. In contrast, with transformation from amastigotes to promastigotes, resistance to meglumine antimoniate was acquired rapidly, within 24 h, before the completion of differentiation. The culture of promastigotes at an acidic pH (5.5) or at an elevated temperature (37 degrees C) alone did not lead to the appearance of SbV susceptibility, emphasizing the requirement of both these environmental factors for the development of SbV susceptibility. A previously isolated sodium stibogluconate (Pentostam)-resistant L. donovani mutant (Ld1S.20) is also resistant to meglumine antimoniate, indicating cross-resistance to SbV-containing compounds. In contrast, no cross-resistance was found with SbIII, suggesting a mechanism of SbV resistance different from that described in Leishmania tarentolae. These data show that L. donovani susceptibility to SbV is parasite intrinsic, stage specific, and macrophage independent.

Cat scratch disease: the rare role of Afipia felis.

Since its isolation in 1988, Afipia felis has been associated with cat scratch disease (CSD) in only one report and its role in CSD has been questioned. We have cultured A. felis from a lymph node of a patient with CSD. 16S rRNA gene sequencing, DNA relatedness studies, fatty acid analysis, and PCR of the A. felis ferredoxin gene showed that the isolate is identical to the previously reported A. felis isolate. To determine the role of A. felis in CSD, PCR of the 16S rRNA gene followed by hybridizations with specific probes were performed with lymph node specimens from CSD patients. All 32 specimens tested positive for Bartonella henselae and negative for A. felis. We conclude that A. felis is a rare cause of CSD. Diagnostic tests not conducive to the identification of A. felis might cause the diagnosis of CSD due to A. felis to be missed.

Major adverse reactions to yeast-derived hepatitis B vaccines--a review.

Yeast-derived recombinant DNA hepatitis B vaccines usage became widely accepted since the early 1990s. Severe adverse events have been reported infrequently in adults and rarely in infants and children given hepatitis B vaccine in the ten years which have passed since the introduction of the vaccine. Some of the data were summarized in previous review articles. Our review of the literature revealed reports of serious adverse reactions which included immediate reactions (anaphylaxis and urticaria) as well as delayed reactions, including skin, rheumatic, vasculitic (including Systemic Lupus Erythematosus and glumerulonephritis), hematologic, ophthalmologic and neurologic reactions. These cases were summarized and a pathogenetic mechanism is offered.

Severe Clostridium difficile-associated colitis in young patients with cystic fibrosis.

We report four patients with cystic fibrosis and fulminant Clostridium difficile-associated colitis: two died, and one required hemicolectomy. Three of four patients carried the N1303K mutation. Severe and fatal C. difficile colitis can occur in cystic fibrosis patients, possibly with a genotype-specific predilection (i.e., N1303K/other). Because cystic fibrosis patients may have a wide spectrum of gastrointestinal symptoms, disease caused by C. difficile must be considered when these patients have acute abdominal pain, diarrhea, or severe leukocytosis.

Molecular diagnosis of cat scratch disease: a two-step approach.

Amplification of Bartonella henselae DNA has been proposed as a diagnostic test for cat scratch disease (CSD). The sensitivities of the following three PCR assays were compared. PCR/rRNA with universal primers amplifies part of the 16S rRNA gene, followed by hybridization with a specific B. henselae probe; PCR/CS and PCR/HSP amplify portions of the gltA and the htrA genes, respectively, each followed by restriction fragment length polymorphism analysis. The threshold of detection of B. henselae DNA in pus was 10(-4), 10(-3), and 10(-2) ng for PCR/rRNA, PCR/CS, and PCR/HSP, respectively. By these three assays, B. henselae DNA was detected in 100, 94, and 69% of 32 pus and lymph node specimens from CSD patients, respectively. The similar sensitivities of the PCR/rRNA and the PCR/CS assays for detecting B. henselae DNA in clinical specimens are in contrast to the 10-fold difference in sensitivities in favor of PCR/rRNA demonstrated with purified B. henselae DNA in sterile pus, suggesting that in the majority of cases, the bacterial load in clinical specimens is large enough to be identified by the PCR/CS assay. A two-step approach is suggested to achieve maximal sensitivity for detecting B. henselae in clinical specimens: initial testing by PCR/CS (which does not require hybridization), followed by PCR/rRNA with PCR/CS-negative specimens when CSD is strongly suspected.

Pentostam induces resistance to antimony and the preservative chlorocresol in Leishmania donovani promastigotes and axenically grown amastigotes.

An axenic amastigote culture system was utilized to directly assess the stage-specific antileishmanial effects of antimony on amastigotes of Leishmania donovani devoid of the macrophage host cell. Pentostam, which contains antimony in the form of sodium stibogluconate and the preservative chlorocresol, was used. Cell density was quantified by measuring the activity of the stable enzyme ornithine decarboxylase. Dose-response curve analyses show that Leishmania promastigotes are susceptible to Pentostam, with the 50% inhibitory concentration (IC50) being 104 microg/ml, while amastigotes are more susceptible, with the IC50 being 24 microg/ml. Promastigotes and amastigotes are also susceptible to chlorocresol, with IC50s being 1.27 and 1.82 microg/ml, respectively. Given that promastigotes are insensitive to antimony, these results suggest that the increased susceptibility of amastigotes to Pentostam is due to the stage-specific activity of sodium stibogluconate. To further study this phenomenon, spontaneous resistance to Pentostam was induced in L. donovani promastigotes by increasing the concentration of Pentostam in the growth medium in a stepwise fashion. Two mutants, Ld1S.04 and Ld1S.20, grew at 0.4 and 2.0 mg of Pentostam per ml, respectively. Promastigotes of these mutants were 11 and 21 times, respectively, more resistant to Pentostam than the wild type. Amastigotes were 40 and 148 times, respectively, more resistant than the wild type. The mutants were also chlorocresol resistant; promastigotes were 6 and 9 times, respectively, more resistant than the wild type, and amastigotes were 14 and 35 times, respectively, more resistant than the wild type. These data show that resistance to Pentostam induced in antimony-insensitive promastigotes is manifested in amastigotes as resistance both to pentavalent antimony and to chlorocresol. The axenic amastigote system is a unique tool which enables direct evaluation of the activity of antileishmanial compounds on the amastigote devoid of its host cell.

Encephalopathy associated with enteroinvasive Escherichia coli 0144:NM infection.

Central nervous system manifestations typically occur with Shigella gastroenteritis and also in enteric Salmonella and Campylobacter infections. To date no association between enteroinvasive Escherichia coli infection and neurologic symptoms has been described. Two children with diarrhea caused by E. coli 0144:NM had otherwise unexplained encephalopathy manifested by profound stupor in one child and by obtundation and meningismus in the other one. These cases of infection occurred in northern Israel during a period of an unusually high rate of enteric infection caused by this organism. None of the microbiologic properties studied were uniquely attributable to the encephalopathic cases. The two encephalopathic as well as all eight nonencephalopathic isolates studied possessed the 140-MDa invasive plasmid. All 10 isolates examined produced small amounts of cytotoxin by the HeLa cell assay, all were nonmotile, and all had identical antibiograms. Eight of 10 of the isolates had identical plasmid profiles, while 2 isolates (from nonencephalopathic patients) had slightly different plasmid profiles. This is the first report of encephalopathy associated with enteroinvasive E. coli.

Can an educational program improve the diagnosis and treatment of pharyngotonsillitis in the ambulatory care setting?

The influence of an educational program on the diagnosis and treatment of pharyngotonsillitis was evaluated in three outpatient clinics in northern Israel during two periods. During both periods--1 January to 31 March 1988 (baseline phase) and 1 January to 31 March 1989 (study phase)--clinical data of all patients for whom antibiotics were prescribed were recorded on special forms, which included the patient's diagnosis and the antibiotic prescribed. In November 1988, 2 months before the study phase, two 1 h sessions on pharyngitis were given by the study physicians to the entire medical staff of two clinics (Clinics B and C), and written material was distributed. A third clinic (Clinic A) served as the control. A comparison of the prescribing habits during the two phases showed that during the study phase the total number of antibiotics prescriptions for pharyngitis declined significantly in Clinics B and C, while the percentage of prescriptions for penicillin V rose with the concomitant decline of amoxycillin. There were no significant changes in prescribing habits in the control clinic. These results show that a modest 2 h educational program involving direct contact with the entire medical staff of the community outpatient clinics can improve the diagnosis of pharyngotonsillitis and reduce both the inappropriate use of antibiotics in general, and the substitution of more expensive antibiotics for cheaper, equally effective ones.