Microfilaria Counts Research Articles

Sensitivity of the nested-polymerase chain reaction (PCR) assay for Brugia malayi and significance of ‘free’ DNA in PCR-based assays

The blood filtration method was used as the gold standard to determine the detection level of simple blood-spot sampling and nested-polymerase chain reaction (PCR) for Brugia malayi. Of 100 samples, 48 were filtration-positive. Of these, 26 had microfilaria counts that were low enough (<1–29 microfilariae/ml) to accurately assess the limit of detection by nested-PCR. Nested-PCR consistently detected B. malayi DNA in samples with ≥10 microfilariae/ml. Post-filtration, microfilaria-depleted, blood-spots from microfilaria-positive samples were screened by nested-PCR and B. malayi specific ‘free’ DNA was detected in 51.7% of these samples. There was no evidence for ‘free’ DNA in microfilaria-negative individuals from this endemic community.

A longitudinal study of Bancroftian filariasis in the Nile Delta of Egypt: baseline data and one-year follow-up.

We initiated a longitudinal study of Bancroftian filariasis to improve understanding of dynamics and risk factors for infection in villages near Cairo, Egypt. Baseline prevalence rates for microfilaremia and filarial antigenemia for 1,853 subjects more than 9 years of age were 7.7% and 11.2%, respectively. Microfilaria counts, antigen levels, and microfilaremia incidence over a 1-year period were all significantly lower in older people. These findings suggest that humans develop partial immunity to Wuchereria bancrofti over time. One-year incidence rates for microfilaremia and antigenemia were 1.8% and 3.1%, respectively. Filarial antigenemia, IgG4 antibody to recombinant antigen BmM14, and household infection were all significant risk factors for microfilaremia incidence. Microfilaria counts and parasite antigen levels were significantly reduced by diethylcarbamazine therapy, but many infected subjects refused treatment, and most treated people were still infected one year later. Incident infections approximately balanced infections lost to produce an apparent state of dynamic equilibrium.

Wuchereria bancrofti antigenaemia in Sri Lanka.

The prevalence of Wuchereria bancrofti antigenaemia determined in 353 subjects in Matara, Sri Lanka by Og4C3 ELISA was 20.7%. Positive rates obtained with the same subjects by 1 ml Nuclepore filtration and 60 microl thick blood smear were 11.3% and 7.9%, respectively. Antigen levels were positively associated with microfilaria counts. Two-thirds of antigen-positive and microfilaria-negative (Ag+/Mf-) individuals were > 25-year-old, but younger age groups (< or = 25-year-old) tended to have proportionally more Ag+/Mf- cases. Possible origins of the Ag+/Mf- status are discussed.

Reduced Loa loa microfilaria count ten to twelve months after a single dose of ivermectin

The action of a single dose of ivermectin (200 μg/kg) on 71 Gabonese patients with Loa loa microfilariae in the peripheral blood, and living in areas highly endemic for loiasis, has been evaluated. Ten to 12 months after treatment, 43 patients (63%) had no circulating microfilaria and the geometric mean peripheral blood microfilaria count had decreased by 88·6% ( P < 0·02).Thus, a single annual dose of ivermectin can markedly reduce loiasis transmission.

Ivermectin for the chemotherapy of bancroftian filariasis: a meta-analysis of the effect of single treatment.

The efficacy and safety of ivermectin in the treatment of filariasis due to Wuchereria bancrofti was assessed by a meta-analysis of the results from 15 published clinical trials. Seven hundred and forty-eight microfilaraemic patients were enrolled in 7 dose-finding and 8 comparative studies. Administered as a single dose, ivermectin induced nearly complete clearance of microfilariae from the blood from the first day to 30 days post-treatment, followed by gradual recurrence of microfilaraemia and increase in its intensity. Higher doses of ivermectin showed greater clearance effects and maintained lower microfilaraemia levels for a longer time. The adverse reactions caused by the drug were flu-like, transient, generally mild and well tolerated by patients. The frequency and intensity of adverse reactions were strongly associated with pretreatment microfilaria counts in the blood, but independent of dose. The findings of the meta-analysis suggest that ivermectin given at a single annual dose of 200 micrograms/kg body weight or higher, whether or not in combination with DEC, has great potential for therapeutic strategies to control bancroftian filariasis.

A polymerase chain reaction-based assay for detection of Wuchereria bancrofti in human blood and Culex pipiens

Human blood samples and indoor-resting Culex pipiens were collected in 33 randomly selected houses from different sectors of a village in the Nile Delta of Egypt which was endemic for Wuchereria bancrofti. Blood was also collected from subjects with no history of living in filarial endemic areas. Human blood samples were divided and assessed by both membrane filtration and polymerase chain reaction (PCR). Similarly, mosquito samples were assessed by both dissection and PCR. Blood pools representing each household were tested by PCR. If a pool gave a positive result, then individual blood specimens were also tested by PCR. Of the 33 houses tested, both membrane filtration and blood pools assayed by PCR identified 14 (42·4%) ‘infected houses’. PCR detected parasite deoxyribonucleic acid (DNA) in blood pools from an additional 3 households that gave negative results by membrane filtration. Of 178 endemic blood samples tested by membrane filtration, 22 (12·3%) had microfilariae and all were individually positive by PCR. Although microfilaria counts were lower in blood collected during the day than in night-collected blood, the PCR results were consistent, regardless of time of collection. All non-endemic blood samples were negative by PCR. Among the 33 houses tested, mosquito pools assayed by PCR identified 17 (51·5%) as ‘infected households’. Of these, 8 houses (47%) contained at least one microfilaraemic resident. One ‘infected household’ was identified by mosquito dissection. We concluded that PCR is a powerful epidemiological tool for screening villages for the prevalence of W. bancrofti. PCR detection of W. bancrofti DNA in blood-fed mosquitoes could be used initially to locate endemic areas with transmission of bancroftian filariasis. PCR detection of W. bancrofti DNA in blood collected during the day could then be used to assess W. bancrofti infection rates.

Control of lymphatic filariasis by annual single-dose diethylcarbamazine treatments

It has long been stressed that diethylcarbamazine citrate must be given at a total dosage of 72 mg per kilogram of body weight in 12 divided doses of 6 mg kg −1 to obtain maximum effect against Wuchereria bancrofti. However, recent studies revealed that only a single dose at 6 mg kg −1 could reduce microfilaria (Mf) counts by 90%, and that the effect would persist for 12–18 months. The annual repeat of the single-dose mass treatment was shown to be effective in reducing Mf prevalence and density in large-scale, long-term field trials. The scheme is simple and economic, and could be sustainable in many endemic areas, where health manpower and resources are often not sufficient. Annual single-dose mass treatments can be an effective weapon against human lymphatic filariasis, as discussed here by Eisaku Kimura and Jona Mataika.

Influence of Anesthetics on the Peripheral Blood Microfilaremia of Brugia malayi in the Mongolian Jird, Meriones unguiculatus

The effects of anesthetics on the peripheral blood microfilaremia of Brugia malayi in Meriones unguiculatus were investigated. Microfilaremias were assessed by orbital puncture prior to and following the use, either individually or in tandem, of ether, Rompun, Ketaset, and sodium pentobarbital. Results indicate that the peripheral microfilaremia varied dramatically, depending on the anesthetic administered. Although microfilaremias were not affected by an initial ether exposure, counts of microfilariae increased significantly when jirds received Rompun and Ketaset, or Ketaset alone. Administration of sodium pentobarbital did not increase the number of microfilariae observed in the peripheral blood. The mode of action differs between these drugs and is likely responsible for the different effects observed. Consequently, studies involving vector-parasite interactions should take precautions to prevent parasite-induced vector mortality due to the ingestion of large numbers of microfilariae induced in the peripheral bloodstream by certain anesthetics.

Irreversible Effects of Ivermectin on Adult Parasites in Onchocerciasis Patients in the Onchocerciasis Control Programme in West Africa

Ivermectin is an effective drug for the treatment of human onchocerciasis, a disease caused by the parasitic filarial nematode Onchocerca volvulus. When humans are treated, the microfilariae normally found in the skin are rapidly and very nearly completely eliminated. Nonetheless, after a delay, microfilariae gradually reappear in the skin. This study is concerned with the causes of this delay. Hypotheses are tested by comparing the results of model calculations with skin microfilaria counts collected from 114 patients during a trial of five annual treatments in the focus area of Asubende, Ghana. The results obtained strongly suggest that annual treatment with ivermectin causes an irreversible decline in microfilariae production of approximately 30%/treatment. This result has important implications for public health strategies designed to eliminate onchocerciasis as a significant health hazard.

Density-dependent processes in the transmission of human onchocerciasis: intensity of microfilariae in the skin and their uptake by the simuliid host.

The transmission success of Onchocerca volvulus is thought to be influenced by a variety of regulatory or density-dependent processes that act at various points in the two-host life-cycle. This paper examines one component of the life-cycle, namely, the ingestion of microfilariae by the simuliid vector, to assess the relationship between intake of larvae and the density of parasites in the skin of the human host. Analysis is based on data from three areas in which onchocerciasis is endemic and includes published information as well as new data collected in field studies. The three areas are: Guatemala (Simulium ochraceum s.l.), West and Central Africa (savanna members of the S. damnosum complex), and South Venezuela (S. guianense). The data record experimental studies of parasite uptake by flies captured in the field and fed to repletion on locally infected subjects who harboured varying intensities of dermal microfilarial infection. Regression analyses of log transformed counts of parasite burdens ingested by the flies plotted against log transformed counts of microfilariae per mg of skin revealed little evidence for saturation in parasite uptake by the flies as the intensity in the human host increased. There was a positive and highly significant rank correlation between both variables for the three blackfly species. In an alternative analysis a model was fitted to data on prevalence of flies with ingested microfilariae (mff) versus dermal mean intensities. The model assumed an overdispersed distribution of the number of mff/fly and a given functional relationship between intake and skin load. The results of both approaches were consistent. It is concluded that parasite ingestion by the vector host is not strongly density dependent in the three geographical areas and ranges of dermal loads examined. It therefore appears that this transmission process is of reduced importance as a regulatory mechanism in the dynamics of parasite population growth.

Clinical and laboratory changes after administration of milbemycin oxime in heartworm-free and heartworm-infected dogs

Summary Adverse reactions to oral administration of milbemycin oxime were investigated in heartworm (hw)-free and hw-infected dogs given either the minimal hw prophylactic dose (0.25 mg/kg of body weight) or the hookworm anthelmintic dose (0.5 mg/kg). In 12 hw-free control dogs treated with lactose excipient (100 mg/kg), abnormal signs were not observed. There were no differences between the 2 doses in prevalence of clinical signs of disease and laboratory test results. In 60 hw-free dogs (50 dogs administered the low dose, and 10 dogs given the high dose) and 46 nonmicrofilaremic hw-infected dogs (35 dogs administered the low dose, and 11 dogs given the high dose), only a transient and slight paleness of the visible mucous membranes, intestinal hyperperistalsis, or both were observed in some dogs. In 77 microfilaremic (mf) dogs (41 dogs administered the low dose, and 36 dogs given the high dose), weakness or loss of appetite was observed in 13 dogs (16.9%). Paleness of the visible mucous membranes was observed in 16 dogs (20.8%), intestinal hyperperistalsis was observed in 27 dogs (35.1%), and respiratory signs, such as mild labored respiration, were observed in 13 dogs (16.9%). Dullness of heart sounds was noticed in 4 dogs (5.2%). In 12 (9 dogs administered the low dose, and 3 dogs given the high dose) of 89 mf dogs (13.5%), adult heartworms migrated from the pulmonary arteries to the right atrium, causing signs of caval syndrome, including heart murmurs, jugular pulsations, and weakness. C and neutrophil counts increased slightly at 3 hours. Serum alanine transaminase, alkaline phosphatasIn hw-free dogs, rectal temperature tended to decrease gradually, and heart and respiratory rates were transiently decreased. Mean blood pressure, rbc count, serum total protein concentration, serum osmolality, and serum sodium (Na) and potassium (K) concentrations decreased 3 or 6 hours after administration. The total WBe, and lactate dehydrogenase activities did not change significantly. In hw-infected, nonmicrofilaremic dogs, trends in laboratory changes were almost the same as those in hw-free dogs. In mf dogs, circulating microfilaria count decreased starting at 6 hours after treatment. Heart rate, rbc count, and serum total protein concentration decreased transiently, similar to values in hw-free dogs. Rectal temperature, respiratory rate, and serum enzyme activities increased, and neutrophil and eosinophil counts, serum osmolality, and serum Na and K concentrations decreased. Changes in test results for dogs with hw migration were almost the same as those in mf hw-infected dogs. Heartworm-free and nonmicrofilaremic hw-infected dogs did not have clinically relevant problems after treatment with milbemycin oxime. Administration of milbemycin oxime does not involve risks for these dogs. However, reactions in mf dogs appear to be related to the death of microfilariae, and caution must be used when these dogs are treated with this compound.

Humoral immune response during filarial fever in Bancroftian filariasis

Humoral immune responses against filarial parasitic infection were studied in 62 cases of acute filarial disease presenting with filarial fever with adenolymphangitis, in a community where Bancroftian filariasis was endemic, during and about one month before and after the febrile episode. Their total leucocyte and differential peripheral blood cell counts and anti-streptolysin O titre were determined and compared. Polymorphonuclear cellular responses and anti-streptolysin O titre did not show any significant alteration during and after fever. Three of 53 previously amicrofilaraemic subjects (9 of whom were initially microfilaraemic) had microfilaria in their circulation during fever, with a significant increase in their geometric mean microfilaria count. Titres of specific immunoglobulin (Ig) G and IgG 4 antibody to Wuchereria bancrofti microfilarial excretory/secretory antigens (measured by enzyme-linked immunosorbent assay) decreased significantly during the fever and the lower levels were maintained one month after fever. The mean circulating immune complex level increased significantly during fever, and a significant percentage of cases demonstrated circulating filarial antigen during fever, which declined after one month, suggesting the release of filarial antigen into the circulation during fever which bound to antibodies to form immune complexes. These observations do not support the suggestion that bacterial infection is the aetiology of filarial fever. It is postulated that antigens released from parasites into the circulation during parturition by adult worms may evoke an allergic response in the host, causing periodic febrile episodes.

Vaccination with recombinant filarial paramyosin induces partial immunity to Brugia malayi infection in jirds.

Vaccination with irradiated infective larvae induces partial protective immunity to infection with the filarial nematode Brugia malayi in jirds. Prior studies have shown that such immunization stimulates a much stronger antibody response to recombinant and native filarial paramyosin than that seen after normal infection. To determine whether vaccination with recombinant paramyosin could induce protective immunity to larval challenge, jirds were immunized with either B. malayi paramyosin and maltose binding protein (BM5-MBP) (fusion protein of B. malayi paramyosin and maltose-binding protein), MBP alone, or recombinant Dirofilaria immitis paramyosin. Animals were challenged with 100 infective larvae s.c. 8 wk after the second immunization. Necropsies were performed 16 wk after challenge. Vaccination with BM5-MBP induced significant protective immunity; adult worm recoveries, worm lengths, and blood microfilaria counts were reduced in the BM5-MBP group relative to the MBP control group. The reductions in adult worm recoveries (43%) and female worm lengths (10%) were statistically significant (p < 0.05). Interestingly, protective immunity was not induced by immunization with D. immitis paramyosin. Additional studies are needed to identify mechanisms involved in protective immunity induced by BM5-MBP and to understand the differential activity of the two closely related recombinant paramyosin proteins in this model.

Transplacental Transmission of Wuchereria bancrofti in Haitian Women

To document the occurrence of transplacental transmission of microfilariae and to determine how frequently it occurred, umbilical cord blood samples and placental tissues were collected from 22 microfilaria-positive women in an area with endemic Wuchereria bancrofti. Microfilaria (mf) counts in the women ranged from 1 to 3,820 mf/ml. Microfilariae were detected in 2 placenta samples and a single cord blood sample. The positive cord blood sample and 1 of the positive placenta samples came from the same woman; no microfilariae were found in a finger prick sample taken from the infant 3 wk after delivery. Our results suggest that microfilariae cross the placenta in less than 10% of pregnancies of microfilaria-positive mothers. Furthermore, the microfilaria count of the mother does not seem to influence directly whether microfilariae are present in the placental blood pool. Although actual transfer of microfilariae to the fetus may occur infrequently, exposure to parasite antigens occurs with much greater frequency. The effect of in utero exposure to either microfilariae or parasite antigens may render newborns tolerant and explain why children born to infected mothers are almost 3 times more likely to become infected than are children born to uninfected women.

Changes in Circulating Parasite Antigen Levels after Treatment of Bancroftian Filariasis with Diethylcarbamazine and Ivermectin

This study assessed changes in circulating parasite antigen levels after diethylcarbamazine (DEC) and ivermectin treatment of bancroftian filariasis to determine effects of these drugs on adult Wuchereria bancrofti in vivo. Thirty adult Haitians with microfilaremia were treated with 1 mg of ivermectin to reduce counts of microfilariae. Later, subjects were treated with either one or two 200 micrograms/kg doses of ivermectin or with 12 daily 6 mg/kg doses of DEC. Macrofilaricidal activity of these drugs was indirectly monitored by measuring circulating W. bancrofti antigen by EIA. Antigen levels fell by 75% after DEC and by 34% after ivermectin. These results suggest that low-dose ivermectin treatment followed by a standard course of DEC is a more effective macrofilaricidal regimen for W. bancrofti than either of the multidose ivermectin regimens used in this study.

Evaluation of a Monoclonal-Antibody Based Antigen Assay for Diagnosis of Wuchereria Bancrofti Infection in Egypt

Conventional methods for diagnosis of Wuchereria bancrofti infection are insensitive and often impractical because of the need for night blood collections. A sensitive and specific antigen detection assay has been developed for W. bancrofti, which is based on a monoclonal antibody (AD12) that binds to a repeated epitope on a 200 kDa adult worm excretion product present in sera from infected humans. The only formal evaluation of this assay to date was performed with sera from India. In the present study, we have evaluated the performance of the AD12 antigen assay in two laboratories with sera collected in endemic and non-endemic areas in Egypt. Antigen was detected in 57 of 59 (97%) sera from microfilaremic subjects, and in 22 of 139 asymptomatic and amicrofilaremic subjects who reside in a highly endemic area. Antigen titers were significantly correlated with microfilaria counts (r = 0.41, P less than 0.01). Filarial antigen was not detected in most sera from amicrofilaremic subjects with clinical filariasis. Comparative antigen test results obtained from laboratories in Cairo and St. Louis agreed in 170 of 173 sera tested. Filarial antigen was not detected in sera from Egyptians with no history of residence in filaria-endemic areas. Specifically, nonendemic sera from patients with other parasitic infections (schistosomiasis, fascioliasis, ascariasis, etc.) were uniformly negative in the assay. We conclude that the AD12 filarial antigen assay is sensitive and specific for W. bancrofti infection in Egypt.

Effect of CGP 20376 on Brugia malayi and Parasite Antigenemia in Jirds

This study was designed to investigate the activity of CGP 20376, a benzothiazole derivative, against Brugia malayi in jirds and to illustrate the utility of parasite antigen detection as a means of monitoring drug efficacy in filariasis. Drug treatment was 100% effective in jirds treated 3 or 24 days after infection. Microfilaria and adult worm counts were reduced (relative to counts in sham-treated control animals) by 96% and 95%, respectively, in animals treated 153 days after infection. Four of 6 animals in this treatment group cleared their microfilaremias and were free of adult worms 5 mo after treatment. Thus, CGP 20376 was effective against all life cycle stages of B. malayi in jirds. Parasite antigen levels in jird sera were consistent with parasitological results in all treatment groups, but antigen clearance was incomplete in some cases after apparently successful treatment of mature and immature infections.

Serological Evaluation of the Macrofilaricidal Effects of Diethylcarbamazine Treatment in Bancroftian filariasis

An Mr 200,000 phosphorylcholine-containing antigen (PC-Ag) of predominantly adult worm origin was found in the sera of humans infected with Wuchereria bancrofti. This paper describes results of a longitudinal study of changes in levels of PC-Ag in response to diethylcarbamazine (DEC) therapy as measured by two-site immunoradiometric assay (IRMA) and Western blotting. One hundred thirty-two residents of a bancroftian filariasis-endemic area of Papua New Guinea (PNG) were treated with a 72 mg/kg dose of DEC. A macrofilaricidal effect was seen with this dose of DEC as 34% of the treated subjects had localized side effects and long-term decreases in microfilariae (mf) counts were observed 12 months after treatment. The PC-Ag levels were reduced to 72%, 52%, and 51% of pretreatment values at 21 days and at six and 12 months after treatment. These decreases, observed by IRMA, were specifically associated with loss of the Mr 200,000 PC-Ag detected by immunoadsorption and Western blotting. From drug treatment data, the maximum half-life of PC-Ag in circulation was calculated to be 50 days, assuming a first-order decay process. This maximum half-life indicates that persistent antigenemia observed in the majority of treated subjects could only result from the survival of adult worms. In the absence of methods to directly demonstrate W. bancrofti adult worms, detection of serum PC-Ag levels provides a sensitive indirect measure of the dynamics of adult worm populations. This serological measurement may be useful in optimizing the macrofilaricidal and therapeutic effects of DEC and in assessing the macrofilaricidal action of new antifilarial drugs and immunological interventions.

Evidence of Nonsusceptibihty to Diethylcarbamazine in Wucheria bancrofti

This study assessed Wuchereria bancrofti-infected patients who received diethylcarbamazine (DEC) to determined if drug levels were comparable between individuals who continued to harbor circulating microfilariae and those whose blood became clear of parasites, between those with high and low microfilaria counts, and between infected and noninfected persons. Haitian volunteers undergoing treatment for W. bancrofti and two nonendemic controls were enrolled. DEC levels in serum and urine samples were determined using a gas chromatographic method. No correlation was found between pre- and posttreatment microfilarial levels and drug levels. Drug levels were comparable in persons with or without residual microfilaria and in infected and uninfected persons. These results indicate that incomplete drug regimens, differences in serum drug levels, and inadequate drug dosage are not the primary causes of treatment failure and suggest there is a degree of parasite tolerance for DEC.

A Comparison of 6-, 12-, and 24-Monthly Dosing with Ivermectin for Treatment of Onchocerciasis

This study was designed to examine the optimal dose and interval of administration of ivermectin, the now-accepted drug of choice for onchocerciasis. Two hundred Liberians with Onchocerca volvulus infection received 100, 150, or 200 micrograms/kg ivermectin or placebo and were followed for 36 months. The reaction after the second dose of ivermectin was significantly less than after the initial dose, although it was still significant in the 200-micrograms/kg group. The skin microfilaria counts in the group treated 6-monthly with 150 micrograms/kg was significantly less than in the group treated yearly (12 and 24 months after initial therapy). Prevalence of microfilariae in the anterior chamber and punctate corneal opacities decreased progressively in all groups over 3 years. There appears to be a slight advantage, in terms of antiparasitic effect over the first 2 years, of therapy given 6-monthly compared with yearly.