Base-pairing of 23 S rRNA ends is essential for ribosomal large subunit assembly.

In ribosomal RNA precursors the spacer sequences bracketing mature 16 S and 23 S rRNA are base-paired to form long helices (processing stems). In pre-23 S rRNA, the processing stem is continued by eight base-pairs of mature 23 S rRNA known as helix 1. Recently, we have found that any part of 23 S rRNA between positions 40 and 2773 could be deleted without the loss of ribosome-like particle formation, while both end regions were indispensable. In this paper we have analyzed the role of the 5' and 3' end regions of 23 S rRNA during ribosomal 50 S assembly in vivo by using mutants of the 23 S rRNA gene. Deletions and substitutions in both strands of the helix 1 lead to the loss of plasmid derived 50 S formation. Compensatory mutations restoring helix 1 were assembled into functional 50 S subunits. We conclude that the helix 1 of 23 S rRNA is the main RNA determinant for ribosomal large-subunit assembly. Deletions in both the 5' and 3' strand of the processing stem reduced the ability of the 23 S rRNA to form ribosomal 50 S subunits. However, even the complete removal of either the 5' or the 3' strand of the processing stem did not abolish the 50 S assembly completely. Thus, processing stem facilitates, but is not essential for assembly.

Analysis of the ribosome large subunit assembly and 23 S rRNA stability in vivo.

The ability of mutant 23 S ribosomal RNA to form particles with proteins of the large ribosomal subunit in vivo was studied. A series of overlapping deletions covering the entire 23 S rRNA, were constructed in the plasmid copy of an E. coli 23 S rRNA gene. The mutant genes were expressed in vivo using an inducible tac promoter. Mutant species of 23 S rRNA, containing deletions between positions 40 and 2773, were incorporated into stable ribonucleoprotein particles. In contrast, if one end of the 23 S rRNA was deleted, the mutant rRNA was unstable and did not form ribosomal particles. Protein composition of the mutant particles was specific; the presence of the primary rRNA-binding proteins corresponded to their known binding sites. Furthermore, several previously unknown ribosomal protein binding sites in 23 S rRNA were identified. Implications of the results on ribosome assembly are discussed.

An A to U transversion at position 1067 of 23 S rRNA from Escherichia coli impairs EF-Tu and EF-G function.

Escherichia coli ribosomes with an A to U transversion at nucleotide 1067 of their 23 S rRNA are impaired in their effective association rate constants (kcat/KM) for both EF-Tu and EF-G binding. In addition, the times that EF-G and EF-Tu spend on the ribosome during elongation are significantly increased by the A to U transversion. The U1067 mutation impairs EF-Tu function more than EF-G function. The increase in the time that EF-Tu remains bound to ribosome is caused, both by a slower rate of GTP-hydrolysis in ternary complex and by a slower EF-Tu.GDP release from the mutated ribosomes. There is, at the same time, no change in ribosomal accuracy for aminoacyl-tRNA recognition. With support from these new data we propose that nucleotide 1067 is part of the ribosomal A-site where it directly interacts with both EF-G and EF-Tu.

Coupling of rRNA transcription and ribosomal assembly in vivo. Formation of active ribosomal subunits in Escherichia coli requires transcription of rRNA genes by host RNA polymerase which cannot be replaced by bacteriophage T7 RNA polymerase.

Transcription of a plasmid-located rrnB operon and the corresponding formation of ribosomes in vivo were studied using either T7 RNA polymerase or host RNA polymerase as transcriptase. The 23 S rRNA gene on the plasmid carried an A1067-->T mutation, which confers resistance against the drug thiostrepton. The proportion of particles containing plasmid-borne 23 S rRNA versus chromosome-borne rRNA was quantified with a precision of better than 10% by scanning sequence autoradiograms around nucleotide 1067. The activity of these particles was determined in the presence of thiostrepton which exclusively abolishes the activity of chromosomal wild-type ribosomes. When the plasmid rrnB operon was transcribed with phage T7 RNA polymerase, up to 80% of the rRNA synthesis was plasmid-directed (pulse labelling) in the late induction phase, most of which (about 85%) became degraded. The cells accumulated 50 S particles with plasmid-borne intact rRNA that was hardly found in 70 S ribosomes, i.e. particles harbouring plasmid-borne rRNA did not enter the pool of active ribosomes. The particles with plasmid-derived rRNAs were also practically inactive in protein synthesis in vitro. However, the rRNA was functional as shown by reconstitution analysis. The same patterns were found at various expression levels of the plasmid rrnB operon, indicating that not the overproduction of rRNA but rather the T7 transcriptase was responsible for the observed effects. However, when the plasmid rrnB operon was transcribed with host RNA polymerase, growth was not affected upon induction, the 30 S to 50 S to 70 S ratios in the cell were not altered, both 50 S subunits and 70 S ribosomes contained large amounts of plasmid-borne rRNA, and the particles with plasmid-derived rRNA were active in vitro. When the induction of rRNA transcription by T7 RNA polymerase was performed at 25 degrees C instead of 37 degrees C, an almost normal pattern was observed. Inactive 50 S particles did not accumulate, and large amounts of plasmid-borne rRNA were found in the pool of 70 S ribosomes. Lowering the induction temperature reduces the transcription rate by T7 RNA polymerase, which is five times faster at 37 degrees C than the host polymerase. The results suggest that the formation of active ribosomal subunits in vivo requires a fine adaptation of the transcription rate of rRNAs and the assembly process, underlining the importance of a coupling between rRNA transcription and ribosome assembly in vivo. T7 RNA polymerase cannot replace the host RNA polymerase in this process at 37 degrees C.

Ribosomal protein L16 binds to the 3'-end of transfer RNA.

Escherichia coli 50 S ribosomal subunits were reconstituted with and without protein L16 present. The latter particles, although active in puromycin reaction, were unable to use CACCA-Phe as an acceptor substrate. We also found that L16 interacts directly with this oligonucleotide and, in the complex with tRNA, protects its 3'-end from pancreatic ribonuclease digestion. We suggest that the role of L16 is in the fixation of the aminoacyl stem of tRNA to the ribosome at its A-site.

The interaction of ribosomal protein L16 and its fragments with tRNA.

Two large proteolytic fragments of Escherichia coli 50 S ribosomal subunit protein L16 were generated by limited hydrolysis with chymotrypsin (missing 9 N-terminal amino acids) and trypsin (missing 16 N-terminal amino acids). It was found that while intact L16 and its chymotryptic fragment both interact with tRNA (Kd = 5.4 x 10(-7) M), the tryptic fragment does not. These results are interpreted in terms of possible significance of the residues 10-16 in the peptidyl transferase activity.

The properties of the complex between ribosomal protein L2 and tRNA.

Escherichia coli ribosomal protein L2 interacts with fMet-tRNAfMet and NacPhe-tRNAPhe in solution, protecting their 3'-ends from enzymatic degradation. At the same time L2 enhances the rate of spontaneous hydrolysis of the ester bonds between terminal riboses and amino acyl moieties of these two peptidyl-tRNA analogues. L2 has, however, only a slight effect on the rate of spontaneous deacylation of aminoacyl-tRNAs. We suggest that the role of L2 is in the fixation of the aminoacyl stem of tRNA to the ribosome at its P-site, and speculate that this protein is directly involved in the peptidyl transferase (PT) reaction.

The force of measles infection in East Africa.

Catalytic models were applied to age-specific incidence data from two East African studies in order to study the force of measles infection in relation to age. The results are compared with the pattern observed in England and Wales. In East Africa the force of measles infection appears to be independent of age, probably as a result of sociocultural factors. Consequently the incidence of measles is highest in the second half of the first year of life and decreases with increasing age. Major vaccination efforts will raise the average age of measles attack, but the peak incidence will remain in the first year of life. This complicates the decision on the optimal age for vaccination and the need for incidence surveillance is stressed. The simple catalytic model, which is based on the assumption of an age-independent force of infection may play an important role in measles surveillance. With this model simple and economic surveillance studies could be designed which use cross-sectional data only.

Toward a framework and indicators for monitoring Roll Back Malaria.

Roll Back Malaria (RBM) is a new global partnership that aims to halve the malaria burden by the year 2010. A framework and indicators for monitoring the outcomes and impact of RBM have been defined through an extensive consultative process. The framework identifies critical areas for monitoring RBM action relating to 1) the impact on malaria burden, 2) improvements in malaria prevention and treatment, 3) related health sector development, and 4) support for RBM action (including partnerships). A set of RBM indicators has been defined that corresponds to these critical areas but that reflects the major variations in malaria epidemiology and the principal interventions in different parts of the world. Countries would select indicators that are appropriate for their situation. Four global indicators are proposed for use by all countries in which RBM action is under way. Data collection procedures are discussed, and it is indicated how monitoring RBM action can build on existing data-collection mechanisms.

The use of grid sampling methodology for rapid assessment of the distribution of bancroftian filariasis.

The recent World Health Assembly Resolution to eliminate lymphatic filariasis as a public health problem once more brings to the fore the need for reliable data for the effective planning of disease control programmes. Most countries do not have data on the distribution of lymphatic filariasis and are therefore not in the position to initiate control programmes based on sound baseline data. We tested in Ghana in 1998-99 a method for the Rapid Assessment of the Geographical Distribution of Bancroftian Filariasis (RAGFIL) that uses a spatial sampling grid with 50 km between sampled villages, rapid assessment surveys for filariasis prevalence in the sampled villages and spatial analysis to estimate the geographical distribution of filariasis throughout the study area. The prevalence contours obtained with the 50 x 50-km sampling grid were operationally similar to those obtained with a 25 x 25-km grid. The predicted prevalence was not statistically different from the sample survey prevalence in 57 independent villages and the 50 x 50-km grid appears adequate for rapid mapping of filariasis. For the purpose of filariasis mapping, the antigen test would seem a better diagnostic test than clinical examination for hydrocoele. We recommend that a regional approach to mapping be used because of the importance of cross-border foci as demonstrated by our findings from the north of Ghana. Application of the method will provide the minimal information required for effective planning of treatment programmes, and will facilitate estimation of the number of people to be treated. It will also help improve estimates of the number of people at risk and affected, and of the burden of disease due to lymphatic filariasis in Africa.

Onchocerciasis infection in children born during 14 years of Simulium control in West Africa.

The incidence of onchocerciasis infection in children born since the start of vector control is one of the indicators used in the epidemiological evaluation of the Onchocerciasis Control Programme in West Africa (OCP). Though initially of littel value, after a decade of control it has become a sensitive indicator of residual transmission. The results of 14 years of control are reported. In 179 villages parasitological surveys were undertaken at intervals of 3-4 years. 15,286 children were examined and 110 were found to be infected, compared to an expected number of 2467 infected had there been no control. There was considerable geographical variation in the results. In the large central OCP area the results were excellent. Of 12,172 children examined in 127 villages, only 23 were found to be infected compared to an expected number of 1960 without control. This suggests that larviciding had achieved a 99% reduction in the incidence of infection in children. Additional surveys in 2 foci in the central OCP area where transmission had relapsed showed that these problems were very localized. Most villages with infected children were found in OCP border areas in the east and west, which had been reinvaded by infective vectors from elsewhere, and in the intermediate area between forest and savanna in Côte d'Ivoire where there had been partial control failures because of resistance. The incidence of infection in children was reduced by an estimated 68% in the eastern reinvaded area, by 87% in the western reinvaded area, and by 84% in the intermediate area.

Changes in ocular onchocerciasis after two rounds of community-based ivermectin treatment in a holo-endemic onchocerciasis focus.

In a longitudinal study to determine the effect of annual community-based treatment of ocular onchocerciasis with ivermectin, the population living in the 3 most affected villages in the holo-endemic onchocerciasis focus of Asubende in Ghana were re-examined 16 and 24 months after initiating treatment. Ocular microfilarial loads had decreased to very low levels in nearly all of the 334 examined persons who were treated twice. Only very few subjects had ocular loads of 32 microfilariae or more in the anterior chamber of the eye, but this was not associated with deterioration of ocular lesions. Important regression of both early and advanced lesions of the anterior segment of the eye was observed, which was highly statistically significant with respect to iridocyclitis. Lesions of the posterior segment of the eye remained stable. Though no systematic change in the visual acuity of the population was observed, 3 new cases of blindness occurred in persons who already had eye lesions at such an advanced stage that ivermectin treatment could no longer affect the outcome. The results suggest that annual ivermectin treatment is adequate to control onchocercal ocular disease even in populations with very high endemicity levels.

Changes in ocular onchocerciasis four and twelve months after community-based treatment with ivermectin in a holoendemic onchocerciasis focus.

The impact of ivermectin mass treatment on ocular onchocerciasis was studied in a holoendemic focus of blinding onchocerciasis in Ghana. A cohort of 417 persons, 369 of whom were treated, was followed up at 4 and 12 months after treatment. The mean ocular microfilarial load in the anterior chamber of the eye and in the cornea of treated persons was reduced to less than 20% and 10% of the pretreatment levels respectively at the 4 months follow-up but had increased significantly by 12 months. Lesions of the eye at the advanced stage of development remained stable. There was significant regression of early lesions of the anterior segment of the eye, particularly iridocyclitis, after ivermectin treatment. In view of the substantial increase of ocular microfilarial loads after 12 months, 6-monthly treatment may be indicated in such highly endemic foci. However, long-term observation is needed to give a correct estimate of the full benefit to be derived from mass treatment with ivermectin.

The impact of five years of annual ivermectin treatment on skin microfilarial loads in the onchocerciasis focus of Asubende, Ghana.

Following the registration of ivermectin (Mectizan) for human use in the treatment of onchocerciasis, in 1987 the Onchocerciasis Control Programme in West Africa (OCP) begun a series of trials in order to determine the safety of the drug when used on a large scale and its potential for morbidity control. This paper reports the changes in skin microfilarial loads during the first 5 years of annual treatment in the holoendemic focus of Asubende in Ghana, which was the largest trial area and which also had the longest series of follow-up surveys. The general observed pattern was a marked reduction of microfilarial loads shortly after each treatment followed by a steady repopulation of the skin until a subsequent treatment round. The overall reduction of microfilarial loads observed between the base line survey and one year after the last treatment was 90% for the total population examined and over 93% for a cohort which received the drug at all 5 treatment rounds. In contrast, there was only a very gradual decrease in the prevalence of infection in the population after subsequent treatments. The study further emphasizes that even a single treatment with ivermectin has a significant medium-term impact on microfilarial loads. Microfilarial counts barely increased after 14-16 months of treatment and stabilized around 55% of pre-treatment counts 2-4 years after a single treatment.

Population dynamics of Onchocerca volvulus after 7 to 8 years of vector control in West Africa.

In an attempt to describe the changing population dynamics of Onchocerca volvulus during a period of vector control, nodulectomies were undertaken in 256 patients from ten villages in the Onchocerciasis Control Programme (OCP) and in 74 patients from two villages in an area with ongoing transmission. A total of 1198 nodules were excised and 4350 adult worms were isolated and examined for viability and productivity. In the OCP villages, the worm population is ageing and dying without replacement by new generations of parasites and various findings signal a breakdown of the worm population after about 12 years interruption of transmission. The sexual activity of the worms was significantly reduced. A Productivity Index was developed to measure the microfilariae production at the nodule level. The reduction in this index for the OCP villages correlates closely with the decline over the control period in the community microfilarial loads in the skin. The results show that it is not only the longevity of the parasite which will determine the duration of vector control, but that the reduced productivity of the ageing parasite population is of equal importance.

Detailed epidemiological mapping of three onchocerciasis foci in West Africa.

Detailed epidemiological mapping of three isolated foci of hyperendemic blinding onchocerciasis was undertaken in three West African countries as part of community trials of ivermectin, a new microfilaricide for the treatment of human onchocerciasis. The results show that the geographical distribution of the prevalence and intensity of onchocerciasis infection in the community can be very different from what was expected on the basis of demographic and entomological information. The technique of detailed epidemiological mapping is an important tool for the identification of target populations for large scale ivermectin treatment of onchocerciasis. It is being used extensively in the Onchocerciasis Control Programme in West Africa.

Distribution and severity of onchocerciasis in southern Benin, Ghana and Togo.

The Onchocerciasis Control Programme in West Africa has recently extended its operation in southern Benin, Ghana and Togo. To estimate the number of people infected and blinded by onchocerciasis and to describe the distribution and severity of the disease in the extension area, 99 villages were selected, using a stratified random sampling procedure, and surveyed. All the ecological and entomological information available was used in the sampling procedure and in the selection of 87 non-representative villages surveyed to confirm the findings. The study estimated that 590,468 people are infected and 11,715 blind from onchocerciasis out of a rural population of 1,878,234. The Pru, Asukawkaw and Mono river basins were areas with high risk of onchocercal blindness. The Oueme and Zou river basins in Benin and the mountainous areas between Ghana and Togo were classified as areas with medium risk of onchocercal blindness. The other parts of the study area presented low or no risk of onchocercal blindness. By detecting the river basins where villagers are at risk of onchocercal disease this study permits the selection of populations for disease control based on mass distribution of ivermectin, a microfilaricide.

The reproductive lifespan of Onchocerca volvulus in West African savanna.

The epidemiological model ONCHOSIM--a model and computer simulation program for the transmission and control of onchocerciasis--has been used to determine the range of plausible values for the reproductive lifespan of Onchocerca volvulus. Model predictions based on different lifespan quantifications were compared with the results of longitudinal skin-snip surveys undertaken in 4 reference villages during 13 to 14 years of successful vector control in the Onchocerciasis Control Programme in West Africa. Good fits between predicted and observed trends in skin microfilarial loads could be obtained for all villages. It is concluded that the reproductive lifespan of the savanna strain of O. volvulus lies between 9 and 11 years, and that 95% of the parasites reach the end of reproduction before the age of 13 to 14 years.

Macrofilaricides and onchocerciasis control, mathematical modelling of the prospects for elimination.

BACKGROUND: In most endemic parts of the world, onchocerciasis (river blindness) control relies, or will soon rely, exclusively on mass treatment with the microfilaricide ivermectin. Worldwide eradication of the parasite by means of this drug is unlikely. Macrofilaricidal drugs are currently being developed for human use. METHODS: We used ONCHOSIM, a microsimulation mathematical model of the dynamics of onchocerciasis transmission, to explore the potentials of a hypothetical macrofilaricidal drug for the elimination of onchocerciasis under different epidemiological conditions, as characterized by previous intervention strategies, vectorial capacity and levels of coverage. RESULTS: With a high vector biting rate and poor coverage, a very effective macrofilaricide would appear to have a substantially higher potential for achieving elimination of the parasite than does ivermectin. CONCLUSIONS: Macrofilaricides have a substantially higher potential for achieving onchocerciasis elimination than ivermectin, but high coverage levels are still key. When these drugs become available, onchocerciasis elimination strategies should be reconsidered. In view of the impact of control efforts preceding the introduction of macrofilaricides on the success of elimination, it is important to sustain current control efforts.

ONCHOSIM: a model and computer simulation program for the transmission and control of onchocerciasis.

ONCHOSIM is a computer program for modelling the transmission and control of the tropical parasitic disease onchocerciasis, or river blindness. It is developed in collaboration with the Onchocerciasis Control Programme in West Africa (OCP), and is used as a tool in the evaluation and planning of control operations. The model comprises a detailed description of the life history of the parasite Onchocerca volvulus and of its transmission from person to person by Simulium flies. The effects of different control strategies, based on larvicide application and chemotherapy (ivermectin), on the transmission and on the disease symptoms can be evaluated and predicted. In the program two simulation techniques are mixed. Stochastic microsimulation is used to calculate the life events of individual persons and inhabitant parasites, while the dynamics of the Simulium population and the development of the parasite in the flies are simulated deterministically. Output of ONCHOSIM conforms to the format in which data collected by the OCP are reported. This enables detailed checking of model specifications against empirical data. Output can also consist of summarizing key indices for the intensity of onchocerciasis infection, which is especially useful for comparing the effectivity of control strategies.