[Reliability evaluation in rural Congo of the calibrated blood smear technique to assess Loa loa microfilaremia level]. / Évaluation en milieu rural au Congo de la fiabilité de la méthode de la goutte épaisse calibrée pour évaluer le niveau de la microfilarémie à Loa loa.

Background-Rationale: The diagnosis of Loa loa microfilaremia consists in the observation, using a microscope, of microfilariae in a sample of peripheral blood spread on a slide and subsequently stained (the "blood smear technique"). The accurate quantification of Loa loa microfilaremia is important because the choice of the first intention treatment depends on the patient's microfilaremia: severe adverse events can occur in individuals with high microfilarial densities when treated with ivermectin or diethylcarbamazine, the latter drug being the only one which can definitively cure the infection. However, despite the widespread usage of this technique and its role in guiding clinical management of the patient, estimates of its reliability remain scarce. Materials and methods: We evaluated the reliability (reproducibility and repeatability) of blood smear technique using several sets of 10 L. loo-positive slides, randomly selected, and considered the results with regard to regulatory requirements. The slides had been prepared as part of a clinical trial conducted in Sibiti, Republic of Congo, a region where loiasis is endemic. Results: The estimated and acceptable coefficients of repeatability (NB: the lower, the better) were 13.6% and 16.0%, respectively. The estimated and acceptable coefficients of intermediate reliability (reproducibility) were 15.1% and 22.5%, respectively. The poorest coefficient of intermediate reliability was 19.5% when the tested parameter was related to the technician who performed the readings (10.7% when the reading day was changed). The inter-technician coefficient of variation assessed using 1876 L. loo-positive slides was 13.2%. The coefficient of inter-technician variation considered acceptable was estimated at 18.6%. Discussion-Conclusion. All estimated coefficients of variability were lower than the calculated acceptable coefficients suggesting reliability of the technique, although the lack of laboratory references precludes any conclusion on the quality of this diagnosis. It is imperative to implement a quality system and standardization of procedures for the diagnosis of L. loo microfilaremia, both in endemic countries and in the rest of the world, where the demand for diagnosis has been increasing for years.

Temporal variability of Loa loa microfilaraemia.

BACKGROUND: The diurnal periodicity of Loa loa microfilaraemia is well known but few studies have documented the short- and long-term stability of microfilarial density. It seems stable over time at the community level, but significant variations have been observed at the individual level. METHODS: We assessed the temporal variability of L. loa microfilaraemia at 5-day, 1-month and 16-month intervals and analyzed the influence of sex, age, level of microfilaraemia, temperatures and time of sampling on this variability. RESULTS: At the community level, L. loa microfilaraemia is very stable over time at 5-day, 1-month and 16-month intervals (Pearson correlation coefficients of 0.92, 0.91 and 0.78, respectively, all three with P < 0.001). However, some individuals had significant variations of up to ± 50% of their initial microfilaraemia at 5-day (33.0%), 1-month (36.5%) and 16-month (62.6%) intervals, even in individuals with an initial microfilaraemia density > 20,000 mf/ml (7.7, 23.1 and 41.4%, respectively, for 5 days, 1 month and 16 months). We do not highlight any external factors that have a major impact on this variability. CONCLUSION: Although at the community level, microfilaria density is very stable, we highlight some individuals with large variations in both the short and long term, which may have an important impact on onchocerciasis control campaigns and longitudinal studies evaluating the impact of an intervention on L. loa microfilaraemia.

Factors associated with the periodicity of Loa loa microfilaremia in the Republic of the Congo.

BACKGROUND: Loa loa microfilariae circulate in the peripheral blood of human hosts following a diurnal periodicity, with maximal microfilaremia levels generally observed between 10:00 am and 3:00 pm. Few studies have assessed factors potentially associated with this periodicity. METHODS: Microfilaremia data were collected repeatedly between 9:00 am and 8:00 pm from 13 individuals in the Republic of the Congo. Using local polynomial regression (LOESS), we determined the best models representing the dynamics of microfilaremia over this period. In a second step, using cosinor models, we evaluated the influence of sex, age, and body temperature on the periodicity of L. loa microfilaremia in blood. RESULTS: All subjects reached their maximum microfilaremia between 10:00 am and 4:00 pm. Individual microfilaremia showed different patterns between individuals, and some clearly showed multiple peaks within a day. LOESS provided a good fit to the observed data. Without adjustment, the maximum microfilarial density was reached around 11:00 am. Adjustment revealed three distinct modes of microfilaremia, occurring around 10:00 am, 1:00 pm, and 4:00 pm. Cosinor models also provided good fit to our data. After adjustment on body temperature, the L. loa microfilaremia fluctuation amplitude decreased significantly from 1684.8 to 310.6 microfilariae(mf)/ml and the predicted peak was estimated at 12:02 pm. CONCLUSIONS: We characterized the periodicity of L. loa microfilaremia mathematically with two different approaches: cosinor models and LOESS regression. Both models suggest that body temperature plays a role in the variation in microfilaremia within a day. Further studies are needed to identify individual co-factors affecting microfilaremia.

Safety and Efficacy of Levamisole in Loiasis: A Randomized, Placebo-controlled, Double-blind Clinical Trial.

BACKGROUND: Individuals with high microfilarial densities (MFDs) of Loa loa are at risk of developing serious adverse events (SAEs) after ivermectin treatment. Pretreatment with drugs progressively reducing Loa MFDs below the risk threshold might help prevent these SAEs. We assessed the safety and efficacy of levamisole for this purpose. METHODS: A double-blind, randomized, placebo-controlled, MFD-ascending trial was conducted in the Republic of the Congo. Participants were treated in 3 cohorts defined by pretreatment MFD and levamisole dose (cohort 1: 1.0kg and 1.5mg/kg; cohorts 2 and 3: 2.5mg/kg). Safety outcomes were occurrence of SAE and adverse event frequency during the first week. The efficacy outcomes were MFD reduction from baseline and proportions of individuals with at least 40% and 80% MFD reduction at day 2 (D2), D7, and D30. RESULTS: The 2 lowest doses (1.0mg/kg and 1.5mg/kg) caused no SAEs but were ineffective. Compared with placebo, 2.5mg/kg levamisole caused more mild adverse events (10/85 vs. 3/85, P=.018), a higher median reduction from baseline to D2 (-12.9% vs. +15.5%, P<.001), D7 (-4.9% vs. +18.7%, P<.001), and D30 (-0.5% vs. +13.5%, P=.036) and a higher percentage of participants with >40% MFD reduction at D2 (17.5% vs. 1.2%, P<.001), D7 (11.8% vs. 6.3%, P=.269), and D30 (18.5% vs. 9.6%, P=.107). CONCLUSIONS: A single 2.5mg/kg levamisole dose induces a promising transient reduction in Loa loa MFDs and should encourage testing different regimens.

Impact of Semi-Annual Albendazole on Lymphatic Filariasis and Soil-Transmitted Helminth Infection: Parasitological Assessment after 14 Rounds of Community Treatment.

Between October 2012 and October 2015, we conducted a community trial to assess the impact of semi-annual (twice yearly) community treatment with albendazole on lymphatic filariasis in Seke Pembe, a village in the Republic of the Congo. Semi-annual community treatment with albendazole has been continued in the community since October 2015. We conducted an additional parasitological assessment survey in October 2019, 6 months after the 14th round of semi-annual treatment. Between October 2012 and October 2015, Wuchereria bancrofti antigenemia and microfilaremia rates in the community had decreased from 17.3% to 4.7% and from 5.3% to 0.3%, respectively. In October 2019, the antigenemia rate had decreased further to 2.8% (19 of 687). No microfilariae were found in night blood smears from persons with circulating filarial antigenemia (0 of 16), suggesting that W. bancrofti transmission has been interrupted in Seke Pembe. Semi-annual albendazole treatments also reduced significantly infection rates with soil-transmitted helminths.

Results From 2 Cohort Studies in Central Africa Show That Clearance of Wuchereria bancrofti Infection After Repeated Rounds of Mass Drug Administration With Albendazole Alone Is Closely Linked to Individual Adherence.

BACKGROUND: Two community trials conducted from 2012 to 2018 in the Republic of Congo and the Democratic Republic of the Congo demonstrated the efficacy of semiannual mass drug administration (MDA) with albendazole (ALB) alone on lymphatic filariasis (LF). However, a high interindividual heterogeneity in the clearance of infection was observed. METHODS: We analyzed trial data to assess the effect of individual adherence to ALB MDA on clearance of circulating filarial antigenemia (CFA) and microfilaremia. Community residents were offered a single dose of ALB every 6 months and tested for LF with a rapid test for CFA at baseline and then annually. CFA test results were scored on a semiquantitative scale. At each round, microfilaremia was assessed in CFA-positive individuals. All CFA-positive individuals for whom at least 1 follow-up measure was available were included in the analyses. Parametric survival models were used to assess the influence of treatment adherence on LF infection indicators. RESULTS: Of 2658 individuals enrolled in the trials, 394 and 129 were eligible for analysis of CFA and microfilaremia clearance, respectively. After adjusting for age, sex, and initial CFA score, the predicted mean time for clearing CFA was shorter in persons who had taken 2 doses of ALB per year (3.9 years) than in persons who had taken 1 or 0 dose (4.4 and 5.3 years; P < .001 for both). A similar pattern was observed for microfilaremia clearance. CONCLUSIONS: These results demonstrate a clear dose-response relationship for the effect of ALB on clearance of CFA and microfilaremia.

The impact of two semiannual treatments with albendazole alone on lymphatic filariasis and soil-transmitted helminth infections: a community-based study in the Republic of Congo.

Implementation of mass drug administration (MDA) with ivermectin plus albendazole (ALB) for lymphatic filariasis (LF) has been delayed in central Africa because of the risk of serious adverse events in subjects with high Loa loa microfilaremia. We conducted a community trial to assess the impact of semiannual MDA with ALB (400 mg) alone on LF and soil-transmitted helminth (STH) infections in the Republic of Congo. Evaluation at 12 months showed that ALB MDA had not significantly reduced Wuchereria bancrofti antigenemia or microfilaria (mf) rates in the community (from 17.3% to 16.6% and from 5.3% to 4.2%, respectively). However, the geometric mean mf count in mf-positive subjects was reduced from 202.2 to 80.9 mf/mL (60% reduction, P = 0.01). The effect of ALB was impressive in 38 subjects who were mf-positive at baseline and retested at 12 months: 37% had total mf clearance, and individual mf densities were reduced by 73.0%. MDA also dramatically reduced the hookworm infection rate in the community from 6.5% to 0.6% (91% reduction), with less impressive effects on Ascaris and Trichuris. These preliminary results suggest that semiannual community MDA with ALB is a promising strategy for controlling LF and STH in areas with coendemic loiasis.

A case study of risk factors for lymphatic filariasis in the Republic of Congo.

BACKGROUND: Little is known regarding risk factors for lymphatic filariasis (LF) in Central Africa. We studied the epidemiology of LF in an endemic village in the Republic of Congo. METHODS: Dependent variables were Wuchereria bancrofti antigenemia (ICT card test) and microfilaremia (night blood smears). The following factors were investigated: sex, age, bed net, latrines, source of water, uptake of anthelmintic drugs, hunting/fishing activities, and occasionally sleeping in the bush. Mixed multivariate logistic regression models were used. RESULTS: 134 of 774 subjects aged ≥ 5 years (17.3%) had W. bancrofti antigenemia and 41 (5.3%) had microfilaremia (mf). Infection rates increased with age up to roughly 20 years and remained stable thereafter. Multivariate analysis of antigenemia demonstrated an increased risk for males (OR = 2.0 [1.3-3.0]) and for people who hunt or fish (OR = 1.5 [1.0-2.4]) and a protective effect of latrines (OR = 0.5 [0.4-0.8]). Among males, those hunting or fishing at night had an increased risk for antigenemia (OR = 1.9 [1.1-3.5]), and use of latrines was protective (OR = 0.5 [0.3-0.9]). For females, bed nets were protective (OR = 0.4 [0.1-0.9]), and there was a strong household effect (intraclass correlation coefficient [ICC]: 0.24). When mf was used as the dependent variable, males had a higher risk for infection (OR = 5.4 [2.1-13.4]), latrines had a protective effect (OR = 0.4 [0.1-0.9]) and there was a marked household effect (ICC = 0.49). CONCLUSIONS: Age, sex, and occupation-dependent exposure to mosquitoes were important risk factors for infection with W. bancrofti in this study. It is likely that men often acquire infection in high transmission areas outside of the village, while children and women are infected in areas with lower transmission inside or near the village. Additional studies are needed to determine whether these findings apply to other areas in Central Africa.

Semi-quantitative scoring of an immunochromatographic test for circulating filarial antigen.

Abstract. The value of a semi-quantitative scoring of the filarial antigen test (Binax Now Filariasis card test, ICT) results was evaluated during a field survey in the Republic of Congo. One hundred and thirty-four (134) of 774 tests (17.3%) were clearly positive and were scored 1, 2, or 3; and 11 (1.4%) had questionable results. Wuchereria bancrofti microfilariae (mf) were detected in 41 of those 133 individuals with an ICT test score ≥ 1 who also had a night blood smear; none of the 11 individuals with questionable ICT results harbored night mf. Cuzick's test showed a significant trend for higher microfilarial densities in groups with higher ICT scores (P < 0.001). The ICT scores were also significantly correlated with blood mf counts. Because filarial antigen levels provide an indication of adult worm infection intensity, our results suggest that semi-quantitative reading of the ICT may be useful for grading the intensity of filarial infections in individuals and populations.

Epidemiology of drug-resistant malaria in Republic of Congo: using molecular evidence for monitoring antimalarial drug resistance combined with assessment of antimalarial drug use.

In Congo, urgent efforts are needed to help with the revision of the national antimalarial drug policy. Despite its high resistance level, chloroquine (CQ) is still extensively used as the first-line treatment for uncomplicated Plasmodium falciparum malaria. The study was conducted in children under 5 years with uncomplicated malaria in Pointe-Noire and Brazzaville, the two largest cities that contain approximately 60% of the population of Congo. We investigated by polymerized chain reaction and sequencing methods the frequency distribution of molecular markers for antimalarial drug resistance, including mutations in P. falciparum chloroquine resistance transporter (pfcrt) gene associated with CQ resistance and mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes conferring resistance to sulphadoxine/pyrimethamine (SP) among pre-treatment P. falciparum isolates, as well as assessing antimalarial drug use in the community. pfcrt (K76T) mutation was present in most isolates (96.4%, n = 138) and high frequency (69.2%, n = 133) of triple-mutant dhfr-S108N, N51I, C59R was observed. The quintuple mutant (dhfr-S108N, N51I, C59R and dhps-A437G or S436A, K540E) considered as molecular marker for SP treatment failure was not found because dhps-K540E mutation was absent in isolates tested; this is a clear evidence for the excellent efficacy of SP that we previously described in the same population. The complete absence of the dhps-K540E mutation is a deterrent component for using this molecular marker as an early warning tool for SP resistance testing in that population. Poor compliance issues related to the antimalarial drug use including inappropriate manufacturing practices reported in this study require intensive attention and should be taken into account when implementing drug policy change. If Congo changes its treatment policy from CQ to SP monotherapy, this will not last long. The strategy of combining SP with other affordable and effective antimalarial drugs such as the artemisinin derivatives to improve efficacy and to delay the development of parasite resistance is essential.

Efficacy of sulfadoxine/pyrimethamine in the treatment of uncomplicated Plasmodium falciparum malaria in Republic of Congo.

Congo is facing frequent failures of treatment of Plasmodium falciparum malaria with chloroquine (CQ), which is still recommended and used as a first-line drug. In Pointe-Noire and Brazzaville, the two largest cities that contain approximately 60% of the population of Congo, we compared the efficacy of CQ versus sulfadoxine/pyrimethamine (SP) for treatment of uncomplicated malaria in children 6-59 months old (mean = 33 months) using the standard World Health Organization (WHO) 14-day in vivo test in two phases between 1999 and 2002. Patients enrolled were randomly assigned to receive SP (25 mg/kg of sulfadoxine and 1.25 mg/kg of pyrimethamine) or CQ (25 mg/kg). In the first phase of the study, 46 patients were assigned to the CQ (n = 23) or SP (n = 23) groups in Pointe-Noire and 52 children were assigned to the CQ (n = 26) or to SP (n = 26) groups in Brazzaville. Results were interpreted according to the WHO lot quality assurance sampling method, and treatment failure rates for SP versus CQ were < 25% versus > 25% in both cities. In the second phase of the study, we accurately determined the actual proportion of treatment failures for SP in Brazzaville. Thus, in 75 of the 80 children enrolled and followed-up until day 14, no clinical or parasitologic failure was recorded and no serious adverse reaction was observed. Since the CQ treatment failure rate exceeds the unacceptable upper limit, SP seems well to be an appropriate alternative for the first-line treatment of uncomplicated P. falciparum malaria, at least in the settings of the present study.