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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bancroftian filariasis is focally endemic in the Nile Delta of Egypt.4 The main vector responsible for transmission of filariasis in Egypt is Culex pipiens L.5 At present, most endemic villages are characterized by low infection prevalence rates, and low infection intensities6 (Egyptian Ministry of Health and Population, unpublished data).
Diethylcarbamazine citrate (DEC) has been widely used for treatment of lymphatic filariasis for more than 50 years.7 The standard regimen recommended for many years (6 mg/kg of body weight per day for 12 days) was difficult to use in large-scale treatment campaigns because of poor compliance. Recent studies have shown that a single, 6 mg/kg dose of DEC is as effective as multi-dose treatment.1,8–11 Single-dose DEC has been recommended as an option for the control of lymphatic filariasis,12,13 and pilot elimination programs in India have used this regimen.
Diethylcarbamazine rapidly reduces MF levels in human blood. Microfilaria levels remain depressed for many months after DEC treatment because the drug kills or temporarily sterilizes adult female filarial worms.14–17 However, DEC does not completely cure filariasis in most cases. Many people remain microfilaremic at low levels after DEC treatment.18 The conventional wisdom is that lightly infected subjects may be able to sustain transmission of filariasis by vector mosquitoes, particularly culicines.19 The foregut barrier, which kills many MF ingested by anophelines, is poorly developed in Cx. pipiens.20,21 In addition, several studies have shown that culicines ingest more MF than expected, based on the volume of blood ingested.22–25 These investigators have emphasized the potential importance of ultra-low density microfilaremia for sustaining filariasis transmission by mosquitoes, but the contribution of such carriers to transmission in the real world is poorly understood.
We have recently reported results of a study of the impact of mass treatment with single-dose DEC in an area of low filariasis endemicity in Egypt.26 We now report results of companion entomologic studies that were performed as part of the single-dose DEC project. The primary goal of these entomologic studies was to examine relationships between MF levels in human blood and Wuchereria bancrofti infection in Cx. pipiens. We also sought to determine whether a minimum threshold density of MF was necessary for these mosquitoes to take up MF and produce third-stage infective larvae (L3). In addition, we studied the effect of single-dose DEC treatment on MF uptake and L3 production by mosquitoes in an attempt to understand how mass treatment with this regimen might affect filariasis transmission.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Source of mosquitoes and experimental feeding on W. bancrofti carriers. Culex pipiens pupae were collected from natural breeding sites in Qalubiya governorate and transported to an indoor insectary with controlled temperature (26 ± 2°C), a relative humidity of 70–80%, and a 16:8 hour photoperiod. Emerging mosquitoes were maintained on a 10% sugar solution until 24 hours before blood feeding. Three to five day-old mosquitoes were transported to the field site for exposure to MF carriers. To coincide with the periodicity of the parasite, blood meals were offered for 30 minutes between 10:00 PM and midnight. Finger prick thick blood films were prepared immediately prior to mosquito feeding for later staining with Giemsa and microscopic examination. Microfilaria levels were defined as the mean number of MF counted in two 50 µL thick smears.
To assess MF uptake by mosquitoes, an aliquot of blood-engorged females was cold-killed immediately after feeding, transported to our laboratory in Cairo, and stored at -70°C. Mosquitoes were later thawed, midguts were dissected out, and ingested blood was lysed in tap water. Blood meals were examined by light microscopy to determine the MF uptake (or ingestion) rate (percentage of engorged mosquitoes with MF) and MF/mosquito. To assess development of L3 in mosquitoes, other blood-fed females were maintained on a sugar solution for 12–13 days (the extrinsic incubation period for W. bancrofti in these mosquitoes in our insectary), dissected, and microscopically examined for the presence and number of L3. The infectivity rate (percentage of mosquitoes with L3) and L3/mosquito were recorded. The L3 yield was calculated for mosquitoes fed on each donor, and defined as L3/mosquito divided by MF/mosquito. Yield was not calculated in cases with no MF ingestion. The MF concentration factor was calculated as described by Lowrie and others.22 These calculations used 2.82 µL as the average volume of blood ingested by Cx. pipiens collected as pupae in the field and raised in an insectary as previously described.23
Assessment of the impact of treatment with single-dose DEC on mosquito vector competence. Microfilaremic subjects were treated with a single oral dose (6 mg/kg of body weight) of DEC (Pharmamed, Ltd., Zejtun, Malta).26 Pre-treatment MF levels for human subjects in this substudy were determined by membrane filtration of 1 mL of venous blood collected at night after 10:00 PM, just before treatment. Mosquito feeding studies were performed as described earlier, 6–7 months following DEC treatment. Eleven of the subjects in the treatment substudy also had mosquito feeding studies performed prior to DEC treatment. Paired data from these subjects were analyzed separately.
Data analysis. Data were analyzed with SPSS 8.0 software (SPSS, Inc., Chicago, IL). Relationships between blood MF levels, MF uptake, and L3 in mosquitoes were assessed by the nonparametric Spearman’s ranked correlation coefficient. The Mann-Whitney U test for nonparametric data was used to analyze differences between two independent group means. Paired data were compared by the nonparametric Wilcoxon signed ranks test. Proportions were tested by the chi-square test. Group comparisons for nonparametric, positively skewed data on MF and L3 per mosquito were performed by analysis of variance with data transformed as square roots.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); many subjects had negative thick smears. Microfilaria counts in thick smears were significantly correlated with earlier MF counts obtained by membrane filtration (rs = 0.63, P < 0.001).
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). Ingestion rates were much higher than infectivity rates in most cases, as were means of MF ingested relative to means of L3 developed. Infected females ingested 1–21 MF and infective mosquitoes contained 1–24 L3.
Relationships between blood microfilaremia and W. bancrofti infection in mosquitoes.
Rates of microfilaria ingestion and development in mosquitoes fed on untreated MF carriers (n = 38) were more closely correlated with MF counts in blood films than in filtered blood (Figure 1). Microfilaria uptake rates were more closely correlated with blood MF counts (by smear or filter) than infectivity rates. Similarly, thick smear MF counts were more highly correlated with the number of MF ingested per mosquito (rs = 0.77, P < 0.001) than with L3 per mosquito (rs = 0.65, P < 0.001). Microfilaria ingestion rates were significantly correlated with infectivity rates (rs = 0.57, P < 0.001), as were the numbers of MF ingested per mosquito, and those of L3 produced (rs =0.48, P = 0.002). The L3 yield (mean ± SD = 0.29 ± 0.38) in mosquitoes that ingested MF from 30 patients did not show a significant correlation with MF counts in smears (rs = 0.10, P = 0.615).
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. The mean ± SD concentration effect was 4.64 ± 4.62 (geometric mean = 3.12).
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and 4). Mosquitoes ingested MF from 100% of smear-positive donors and 88.2% of these feedings produced L3. Smear-negative/filter-positive MF carriers were relatively poor sources of MF compared with smear-positive carriers, although mosquitoes ingested MF from 47.6% of smear-negative donors and produced L3 following 33.3% of these feeds. These percentages were significantly lower than those observed in mosquitoes fed on donors with positive smears (P < 0.001 for both comparisons by chi-square test). Rates of MF uptake and infectivity for mosquitoes fed on smear-negative MF carriers were significantly lower than those observed for mosquitoes fed on smear-positive donors (P < 0.001 for both comparisons by Mann-Whitney U test). Intensities of infection (MF/mosquito and L3/mosquito) were also much lower in mosquitoes fed on filter-positive/smear-negative subjects than in those fed on smear-positive ones. Reductions in these parameters were 86.3 and 93.1%, respectively (P < 0.001 for both comparisons).
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, with data on MF ingestion and L3 production. As in untreated subjects, MF counts were more closely correlated with MF ingestion (rs = 0.89, P < 0.001) than infectivity (rs = 0.61, P = 0.001), and MF uptake rates were, again, moderately correlated with infectivity rates (rs = 0.61, P = 0.001).
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), and separately for the subset of MF carriers that had mosquito feeding performed both before and after treatment (Table 5).
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) were much lower than those observed for untreated smear-negative MF carriers, but these differences were not statistically significant. Female mosquitoes fed on subjects who were MF smear-negative after treatment contained significantly fewer MF/mosquito (94.1% fewer) and fewer L3/mosquito (50% fewer) than those fed on untreated smear-negative subjects. The L3 yield by mosquitoes fed on three treated smear-negative subjects with MF uptake > 0 (see Table 3) was not significantly different from that observed for 10 untreated smear-negative subjects with MF uptake > 0 (see Tables 1 and 4) (P = 0.215).
Smear-positive treated subjects.
Microfilaria counts by thick smear at the time of mosquito feeding on untreated and treated, smear-positive human subjects were comparable (mean ± SD = 7.2 ± 6.4 and 8.4 ± 10.9 MF/50 µL of blood, respectively; P = 0.776). Mosquitoes ingested MF from all human hosts in both smear-positive groups. Interestingly, mosquitoes fed on treated donors had significantly lower MF uptake rates compared with mosquitoes fed on untreated subjects (Table 4). Females fed on treated subjects also contained significantly fewer MF/mosquito (59.7% reduction). Mosquitoes produced L3 after feeding on 12 (80.0%) of 15 treated smear-positive donors. This rate was not significantly different from that observed before treatment in smear-positive subjects (P = 0.645). Infectivity rates were comparable for mosquitoes fed on these two donor groups. However, L3/mosquito was significantly lower (24.1% reduction) in the treated smear-positive group. This is consistent with the decreased MF/mosquito value observed for females fed on these subjects. The L3 yield in mosquitoes fed on 15 treated smear-positive subjects was not significantly different from that observed for 17 untreated smear-positive subjects (P = 0.298).
Subjects tested before and after treatment.
Eleven subjects had mosquito feeding studies performed before and 6–7 months after single-dose DEC treatment. These subjects have host code numbers < 39 (listed in Tables 1 and 3). Results obtained from these subjects are shown in Table 5. Microfilaria counts by thick smear were reduced by 87.9% after treatment, and seven of 10 subjects had complete MF clearance by smear. Before treatment, mosquitoes ingested MF from all of these subjects and produced L3 from 10 of them. After treatment, females ingested MF from six of 11 subjects; four of these feedings produced L3. Microfilaria ingestion rates and MF/mosquito decreased significantly following treatment. Likewise, infectivity was greatly reduced in eight of 10 cases, whereas it paradoxically increased by 20% and 123.3%, respectively, in two cases. These cases prevented decreases in infectivity from being statistically significant. If these outliers are excluded from the analysis, infectivity decreases from 13.3 ± 19.7 to 0.4 ± 0.9 (mean ± SD) (P = 0.012) following DEC treatment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methods used in this study differed from those used in some prior studies of MF uptake by mosquitoes. For example, we focused on subjects with low MF counts to better understand parasite uptake in situations with low infection intensities that would be expected in later phases of elimination programs. Also, we exposed female mosquitoes that had emerged from field-collected pupae to the forearms of microfilaremic volunteers,27 and mosquitoes were fed at the optimal time for MF uptake based on the periodicity of the local parasite. Other studies used colonized mosquitoes,24,28–30 or exposed mosquitoes to volunteers sleeping under bed nets.22,31 We believe that the latter method has clear disadvantages when compared with our method. With bed nets, microfilaremia cannot be assessed at the exact time of feeding and mosquitoes are permitted to sample blood from different areas with varying MF densities.22
We found that MF uptake and infectivity in mosquitoes were more closely related to MF counts in finger prick blood (assessed by thick blood smears) than in venous blood (assessed by membrane filtration). This was not surprising since mosquitoes feed directly on capillary blood, which tends to contain more MF than an equal volume of venous blood.32,33 The fact that smears were from the night of feeding while filter data were from several months prior to feeding also may have contributed to this stronger correlation. Infectivity rates and L3/mosquito were less closely correlated with blood microfilaremia than MF uptake. This reflected our observation that only approximately one-third of the MF taken up by Cx. pipiens matured to the infective stage. It is interesting that L3 per mosquito exceeded MF per mosquito in several cases (Tables 1 and 3). This is likely due to sampling error, since fewer mosquitoes were dissected to assess MF uptake to maximize our chances of detecting L3.
Prior studies have attempted to define absolute MF density thresholds below which filariasis transmission would be interrupted. For example, Shaoqing and others34 proposed a threshold level of 12 MF/60 µL of capillary blood for transmission of Brugia malayi by anophelines. Other studies suggest that the density threshold is much lower for culicine-transmitted filariasis, presumably because of anatomic differences between these two groups of mosquitoes.20,21 For example, a study of bancroftian filariasis in Sri Lanka showed that carriers with fewer than 10 MF/ml were able to infect Cx. quinquefasciatus.35 On the other hand, Sabry33 (working in Egypt) discounted the importance of low-density carriers with fewer than 15 MF/ml or 2 MF/20 µL of finger prick blood. We did not observe an absolute threshold blood MF level below which mosquitoes failed to ingest MF. Indeed, mosquitoes fed on smear-negative infected volunteers ingested at least one MF in almost half of the feedings and produced at least one L3 in one-third of the cases. Our results also confirmed prior observations that Culex mosquitoes ingest more MF than expected, based on the size of a blood meal.22–25 This finding supports the potential value of collecting fed mosquitoes resting within houses for detecting filariasis in areas of low endemicity and also for monitoring changes in transmission following implementation of elimination programs.36,37
It is important to emphasize that although MF were sometimes taken up by Culex mosquitoes fed under controlled conditions on smear-negative MF carriers (and recognizing that some of these MF developed to L3 in the insectary), such low- and ultra-low level MF carriers are unlikely to sustain filariasis transmission under natural conditions. Microfilaria uptake and infectivity in mosquitoes fed on smear-negative subjects were much lower than those observed in mosquitoes fed on smear-positive carriers with low to moderate MF counts (range = 1–20 MF/50 µL of smear). Also (as mentioned earlier), a majority of ingested MF failed to develop to the L3 stage. When combined with other considerations, we believe that the contribution of smear-negative carriers to culicine-transmitted filariasis is negligible, and for practical purposes can be ignored. Consider the following: For a mosquito to transmit filariasis, it must ingest MF from an infected human, be permissive for parasite development, survive the extrinsic incubation period of the parasite, and transmit L3 during a subsequent feeding on a human. Unpublished observations by our group have shown that only about 60% of blood fed Cx. pipiens captured in houses in endemic villages in the Nile delta contain human blood, and no more than 3% of the mosquitoes in these areas survived the extrinsic incubation period of the parasite (approximately 13 days in Egypt) during any month of the year. Not all L3 leave the mosquito during feeding, and many L3 deposited on the skin fail to enter the mosquito’s puncture wound and gain entrance to the human body.38,39 The proportion of entering L3 that survive to become adult worms is unknown, but this must be much less than 100%. Taken together, these factors explain the well-known inefficiency of filariasis transmission.19,40
Although we have used Egyptian examples to make this case, we believe that our conclusions about inefficient transmission are applicable in many filariasis-endemic areas. This is the Achilles heal of the parasite, and the theoretical basis for the global elimination program. Our results suggest a clear target for filariasis elimination programs, at least for areas with culicine transmission; we believe that one major goal of filariasis elimination programs should be to reduce MF prevalence rates by 50 µL thick blood smears to zero. While a negative smear is not an absolute threshold for MF uptake, we believe that this is a functional threshold in terms of transmission and a practical target for elimination programs.
If residual infections with MF detectable only by membrane filtration are unlikely to sustain transmission, it follows that there should be no need to expend resources to detect ultra-low level MF carriers in populations by membrane filtration if MF prevalence rates are zero by thick smear. A zero MF prevalence rate by thick smear would be one good indication that transmission has been interrupted. Mosquito monitoring would be useful to confirm this finding. Note that other tools are needed to determine the time when elimination efforts (mass treatment) can safely be stopped in the course of elimination programs. Our group is exploring the use of antigen testing of population samples and antibody testing in young children for this purpose in Egypt.
The second major objective of our study was to look at effects of single-dose DEC treatment on infection parameters in mosquitoes. To do this, we compared results of mosquito feeding studies that were performed on treated and untreated volunteers who had comparable blood MF counts. The purpose of this substudy was to determine whether MF remaining in human blood after single-dose DEC treatment are fully functional and capable of sustaining transmission by mosquitoes. It is important to note that MF incidence rates in the study villages were documented to be less than 1% per year in the years just prior to the present study6 (also, authors’ unpublished data). Thus, MF present in blood six months after treatment were not likely to have resulted from new infections.
Six months after treatment with single-dose DEC, a considerable proportion of treated, smear-negative patients were still capable of infecting mosquitoes. One of the infective patients was amicrofilaremic by filter and smear (but still antigen-positive). Parallel, unpublished studies carried out by our group have revealed that mosquitoes that fed on antigen-positive/filter-negative subjects ingested MF in eight (11.1%) of 72 cases. Again, these feedings produced few L3, and it is unlikely that such subjects contribute significantly to transmission. However, prior studies by our group have shown that antigen-positive, amicrofilaremic subjects are at increased risk of developing microfilaremia over the following year.6 These considerations underline the importance for elimination programs to treat all people in endemic areas (i.e., not only those with detectable MF) and to continue mass treatment for several years.
Many of the treated subjects in our study had persistent microfilaremia 6–7 months after single-dose DEC. Other groups have reported that patients with high MF densities pre-treatment often fail to clear MF after DEC treatment.41,42 Such residual MF have been assumed to be fully functional, but this has not been carefully studied in the past. We were surprised to find that mosquitoes ingested fewer MF from subjects with persistent microfilaremia following treatment than from untreated MF carriers with equivalent blood MF counts. We do not fully understand this finding; it is possible that a subset of the persistent MF are damaged in such a way that they are not taken up well by mosquitoes. On the other hand, MF ingested by mosquitoes from treated subjects matured normally to become L3. Thus, MF carriers that remained smear-positive after single-dose DEC treatment represent a potentially significant source of infection to mosquitoes. This finding underlines again the importance for filariasis elimination programs to continue mass treatment activities to achieve the goal of reducing MF smear rates to zero.
We also studied a group of subjects before and after they were treated with DEC to more directly assess effects of treatment on MF levels and mosquito infection parameters. Paired pre-treatment and post-treatment MF and mosquito data were only available for a relatively small number of subjects (n = 11). However, the only prior study like this reported results for two subjects.30 Single-dose DEC was highly effective in our subjects, with reductions in blood MF levels similar to those reported after single-dose DEC in Samoa and French Polynesia17,43 and after multidose (72 mg/kg) DEC in India.44 Paired mosquito feeding data showed that the ability of these subjects to infect mosquitoes was greatly reduced 6–7 months after single-dose DEC. Small sample size (with two outliers) prevented the decreases in infectivity from being statistically significant, although the change in L3/mosquito was highly significant.
To conclude the treatment portion of the discussion, we found that single-dose DEC had a dramatic impact on MF uptake and L3 production in our subjects and that residual MF present in blood 6–7 months after treatment were capable of developing to the infective L3 stage.
Our study was performed in an area with low filariasis prevalence rates and low baseline infection intensities. Our results suggest that mass treatment with repeated cycles of DEC (without albendazole) might be sufficient to interrupt transmission in such areas. We believe that parallel studies of MF uptake and maturation should be performed in a variety of areas with different levels of endemicity, vector species, and epidemiology. Such studies would provide important empirical data for planning and assessing filariasis elimination programs.
Received August 8, 2002. Accepted for publication December 5, 2002.
Acknowledgments: We thank the Entomology Field Team of the Research and Training Center on Vectors of Diseases at Ain Shams University for excellent technical assistance.
Financial support: This study was supported by National Institutes of Health grant NO1 UO1 AI-35855.
Authors’ addresses: Hoda A. Farid, Ragaa E. Hammad, Doaa A. Soliman, Maged A. El Setouhy, and Reda M. R. Ramzy, Research and Training Center on Vectors of Diseases, Faculty of Science Building, Ain Shams University, Abbassia, Cairo, Egypt. Gary J. Weil, Infectious Diseases Division, Barnes-Jewish Hospital, 216 S. Kingshighway, St. Louis, MO 63110.
Reprint requests: Gary J. Weil, Infectious Diseases Division, Box 8051, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, Telephone: 314-454-7782, Fax: 314-454-5293, E-mail: gweil{at}im.wustl.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
0.001 for both). Correlation coefficients for blood MF counts by membrane filtration versus MF ingestion and infectivity rates were 0.61 and 0.52, respectively (P