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Background: Compared with women who are human immunodeficiency virus (HIV) negative, women with human immunodeficiency virus (WWH) have a higher human papillomavirus (HPV) prevalence and increased cervical cancer risk, emphasizing the need for effective cervical cancer screening in this population. The present study aimed to validate methylation markers ASCL1 and LHX8 for primary screening in a South African cohort of WWH., Methods: In this post hoc analysis within the DIAgnosis in Vaccine And Cervical Cancer Screen (DiaVACCS) study, a South African observational multicenter cohort study, cervical scrape samples from 411 HIV-positive women were analyzed for hypermethylation of ASCL1 and LHX8 genes, HPV DNA, and cytology. Sensitivities, specificities, and positive and negative predictive values of primary methylation-based, HPV-based and cytology-based screening were calculated for the detection of cervical intraepithelial neoplasia of grade 3 or higher., Results: Single markers ASCL1 and LHX8 resulted in a good performance for the detection of cervical intraepithelial neoplasia of grade 3 or higher, with sensitivities of 85.9% (95% confidence interval [CI], 78.2%-93.6%) and 89.7% (83.0%-96.5%), respectively, and specificities of 72.9% (67.3%-78.5%) and 75.0% (69.5%-80.5%). Combining markers ASCL1 and LHX8 resulted in a lower sensitivity compared with HPV testing (84.6% vs 93.6%, respectively; ratio, 0.90 [95% CI, .82-.99]) and a higher specificity (86.7% vs 78.3%; ratio 1.11 [1.02-1.20]) and reduced the referral rate from 46.8% to 33.4%. ASCL1/LHX8 methylation had a significantly higher sensitivity than cytology (threshold, high-grade intraepithelial squamous lesion or worse), (84.6% vs 74.0%, respectively; ratio, 1.16 [95% CI, 1.01-1.32]) and similar specificity (86.7% vs 91.0%; ratio, 0.95 [.90-1.003])., Conclusions: Our results validate the accuracy of ASCL1/LHX8 methylation analysis for primary screening in WWH, which offers a full-molecular alternative to cytology- or HPV-based screening, without the need for additional triage testing., Competing Interests: Potential conflicts of interest. C. J. L. M. M., and R. D. M. S. are minority shareholders of Self-screen, a spin-off company of Amsterdam UMC, location Vrije Universiteit; Self-screen develops, manufactures and licenses high-risk HPV and methylation marker assays for cervical cancer screening and holds patents on these tests. A research use only version of the methylation assay has been made available by Self-screen (www.self-screen.nl). C. J. L. M. M. is the part-time chief executive officer of Self-screen and served occasionally on scientific advisory boards and/or speakers bureaus for GlaxoSmithKline (GSK), Qiagen, Sanofi Pasteur MSD/Merck, and Asieris Pharma/Ismar Healthcare. He also reports consulting fees from Qiagen, GSK, Sanofi Pasteur MSD/Merck, Asieris Pharma/Ismar Healthcare, paid to the author, and support for attending meetings and/or travel, paid to the author, from Qiagen, GSK, Sanofi Pasteur MSD/Merck, and Self-screen. He formerly had a very small number of shares of MDXHealth and Qiagen. R. D. M. S. is an inventor on patents on methylation markers for cervical cancer (patents are licensed to Self-screen). All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Although diethylcarbamazine (DEC) and albendazole are recommended to interrupt transmission of Wuchereria bancrofti, little is known about the macrofilaricidal effect of this drug combination. Forty-seven men with W. bancrofti infection were randomly assigned to receive a single dose of either DEC alone (6 mg/kg) (n=25) or a combination of DEC (6 mg/kg) and albendazole (400 mg) (n=22). Physical examinations for scrotal nodules (resulting from worm death) and ultrasound examinations (to detect living adult worms) were performed before treatment and 7, 14, 30, 45, 60, 90, 180, 270 and 360 days after treatment. Blood was examined for microfilariae before and 30 days and 360 days after treatment. Seven days post treatment, intrascrotal nodules were detected at the site of 21 (46.7%) adult worm nests in men who received DEC alone compared with 2 (6.1%) sites in men who received DEC and albendazole (P=0.002). One year after treatment, 10 (22.2%) original adult worm nests remained detectable by ultrasound among men who received DEC alone compared with 18/32 (56.3%) nests among men who received both drugs (P=0.016). Microfilaraemia prevalence and density decreased to a similar extent in both groups. Addition of albendazole appeared to decrease the macrofilaricidal effect of DEC against W. bancrofti, with no detectable enhancement in microfilarial suppression.

To interrupt transmission of Wuchereria bancrofti, a parasite that causes lymphatic filariasis, mass treatment of at-risk populations with antifilarial drugs is recommended for 4-6 years, the minimum estimated adult worm lifespan. Factors associated with adult worm longevity are unknown. In Recife, Brazil, we conducted a retrospective cohort study of 57 men whose adult W. bancrofti were not sensitive to diethylcarbamazine and who were followed with semi-annual physical examinations (to detect intrascrotal nodules, indicative of adult worm death) and ultrasound examinations (to detect the 'filaria dance sign' (FDS), indicative of living adult worms). After 5 years, the FDS remained detectable in 10 (24.4%) of 41 adult worm nests in 25 men from areas of high filariasis transmission intensity and in 30 (90.9%) of 33 nests in 32 men from areas of low transmission (P<0.001). New nodules and adult worm nests were detected only in men from high-transmission areas. Of 30 men who were microfilaria-positive initially and whose FDS remained detectable after 5 years of follow-up, 19 (63.3%) remained microfilaria-positive in 5 ml blood (mean density, 0.4 per ml). In conclusion, survival of adult W. bancrofti is inversely associated with transmission intensity. These findings have implications for filariasis elimination and research.

Although testicular hydrocele is the most common clinical manifestation of bancroftian filariasis, its pathogenesis is poorly understood, as is its relationship to inflammatory scrotal nodules following death of adult Wuchereria bancrofti. Between 1994 and 1998, we prospectively determined the incidence and clinical evolution of nodule-associated acute hydrocele in men attending 2 outpatient clinics in Recife, Brazil who were infected with W. bancrofti, had living adult worms detectable by ultrasound in the intrascrotal lymphatic vessels, and were scheduled for treatment with 6 mg/kg diethylcarbamazine (DEC). A total of 132 men developed 173 scrotal nodules 1-7 (mean 4.2) d after DEC treatment and another 47 developed 58 spontaneous nodules before they received DEC treatment. These 179 men with a single 'nodule event' (simultaneous development of > or =1 scrotal nodules) were followed-up by serial physical and ultrasound examinations for 18 months. Overall, 40 (22.3%) men developed acute hydrocele, 3 of whom underwent biopsy and hydrocele repair. Of the remaining 37 men, 9 (24.3%) developed chronic hydrocele and 28 had acute hydrocele resolution within 14-210 (mean 60.9) d. Rate of chronic hydrocele was similar for men who received DEC and those with spontaneous nodules. Seventeen (42.5%) men with hydrocele had multiple scrotal nodules, compared with 28 (20.1%) men who did not develop hydrocele (P= 0.007). Of 134 men with single nodules, superior paratesticular nodules were found in 56.5% and 29.7% of those with and without hydrocele, respectively (P = 0.02). Acute hydrocele occurs frequently following death of adult W. bancrofti and single episodes of scrotal nodule formation. Chronic hydrocele may develop following 5.1% of these episodes.

Bancroftian filariasis, a mosquito-transmitted disease commonly known as elephantiasis, is caused by infection with the parasite Wuchereria bancrofti. Infection with this parasite can induce a broad array of chronic debilitating and socially stigmatizing conditions, but the pathogenesis of this morbidity remains obscure. Recent evidence indicates that in filariasis-endemic areas the primary lesion is not lymphatic vessel obstruction but, rather, dilatation. To determine the extent to which lymphatic dilatation occurs in the presence of living adult W. bancrofti, we performed longitudinal ultrasonographic measurements in 80 men (mean age 24 years) in Brazil who had a total of 107 W. bancrofti nests detectable by ultrasound. Initial mean lymphatic vessel diameter at the site of the worms was 3.4 mm (range, 0.7-11.3), and was greater in men with 2 or more nests (3.9 mm) than in those with only one nest (3.0 mm, P = 0.003). During the study period (2-35 months, mean, 13.7), lymphatic vessel diameter increased at the site of 92 (86.0%) adult worm nests. Mean rate of increase of lymphatic vessel diameter was 1.2 mm per person-year (range, 0-0.93 mm per month). In a general linear model, no factors, including treatment with antifilarial drugs, were significantly associated with rate of vessel diameter increase. Thus, lymphatic vessel dilatation progress in the presence of living adult W. bancrofti; the rate of this progression is heterogeneous. These data suggest that lymphatic dilatation will continue to progress in most infected persons even after mass treatment with currently recommended antifilarial drugs. In addition to interrupting transmission, the global programme for elimination of lymphatic filariasis should address the potential for disease progression in persons who remain infected with adult W. bancrofti.

The value of the polymerase chain reaction (PCR) in the diagnosis of Wuchereria bancrofti infection was evaluated in comparison to microscopical examination of night blood smears, Nuclepore filtration, serology and ultrasonography. No correlation was found between PCR-based deoxyribonucleic acid (DNA) probing and serology. We did not find any evidence of free filarial DNA in either blood plasma or chylocoele fluid. We conclude that the 2 PCR-based techniques evaluated are not more sensitive than Nuclepore filtration for detection of W. bancrofti microfilaraemia, need at least 1 intact microfilaria in the volume of blood used for DNA extraction, and were much inferior to ultrasonography for detection of amicrofilaraemic adult worm carriers.

Little is known about lymphatic filariasis or the anatomical location of adult Wuchereria bancrofti in children. Seventy-eight children from Greater Recife, 23 microfilaria-positive and 55 microfilaria-negative in approximately 60 microL blood, underwent ultrasound examinations of the major superficial lymphatic vessels of the limbs, scrotal area (boys), and breast area (girls). The characteristic movements of adult worms, known as the filaria dance sign (FDS), were detected in 11 (14.1%) children. In 9 boys, the FDS was detected in lymphatic vessels of the scrotal area (8, ages 14-16) and the inguinal cord (1, age 11). In girls, the FDS was detected in a crural lymphatic vessel and an axillary lymph node. FDS detection was more common in boys (P = 0.06), older children (P = 0.001), and children with microfilaraemia (P = 0.05). Diffuse lymphangiectasia was visualized in 4 boys (ages 14-16) and 2 children had clinical signs of filariasis. These ultrasonographic findings associate W. bancrofti with both infection and disease in children.

The natural history of lymphatic disease in human filariasis remains unclear, but recurrent episodes of acute lymphangitis are believed to constitute a major risk factor for the development of chronic lymphoedema and elephantiasis. Prospective analysis of 600 patients referred to the filariasis clinic of the Centro de Pesquisas Aggeu Magalhães/FIOCRUZ in Recife, Brazil, indicated that 2 distinct acute syndromes accompanied by lymphangitis occur in residents of this filariasis-endemic area. One syndrome, which we call acute filarial lymphangitis (AFL), is caused by the death of adult worms. It is relatively uncommon in untreated persons, usually is asymptomatic or has a mild clinical course, and rarely causes residual lymphoedema. The second syndrome, of acute dermatolymphangioadenitis (ADLA), is not caused by filarial worms per se, but probably results from secondary bacterial infections. ADLA is a common cause of chronic lymphoedema and elephantiasis in Recife as well as in other areas of Brazil where lymphatic filariasis is not present. The syndromes of AFL and ADLA can be readily distinguished from each other by simple clinical criteria.

Studies using conventional angiography or non-invasive scintigraphy have revealed widespread abnormalities in the lymphatics of the legs of patients with bancroftian filariasis, regardless of whether clinical lymphoedema is present. To determine if the observed changes were specific for filarial infections, we imaged the lymphatics of both legs in native residents of an area in Brazil where filariasis is not endemic. Study participants were matched by age, socioeconomic status and physical activities to patients with filariasis in Recife, evaluated in parallel. Based on textbook criteria, only one of 15 study participants had a completely normal lymphoscintigram. Modest to severe pathology of the leg lymphatics was observed in the remaining 14 residents of the non-endemic area and in 49 of 50 patients with bancroftian filariasis. These results indicated that factors other than filarial worms are a common cause of subclinical pathology of the leg lymphatics in north-eastern Brazil, and that the latter is not specific for bancroftian filariasis.

When ivermectin and diethylcarbamazine (DEC) are given simultaneously in a single dose to persons with Wuchereria bancrofti infection, the resulting suppression of microfilaraemia is more profound and sustained than when either drug is given alone. To assess whether this effect is a result of enhanced macrofilaricidal efficacy, we used ultrasound to monitor the adult worms in the scrotal area of men with W. bancrofti microfilaraemia. Twenty-one men were treated simultaneously with DEC (6 mg/kg) and either 200 micrograms/kg or 400 micrograms/kg of ivermectin (11 and 10 men, respectively). Ten other men received a single 200 micrograms/kg dose of ivermectin followed 5 d later by a 6 mg/kg dose of DEC (sequential treatment). All men became amicrofilaraemic after treatment and all except one remained so for one year. Cessation of adult worm movement, indicative of death of all the adult worms in a given 'nest', was observed in none of 30 nests in men who received simultaneous treatment and in 3 of the 19 nests (16%) in the men who received sequential treatment (P = 0.05). Scrotal nodules were detected in 5 of 21 men (24%) who received simultaneous treatment and in 8 men (80%) who received sequential treatment (P < 0.01). Thus, co-administration of ivermectin with DEC seems to interfere with the macrofilaricidal action of DEC. These findings have implications both for treatment of the individual patient and for community-based drug distribution programmes designed to interrupt transmission of W. bancrofti.

To assess directly the effect of various doses of diethylcarbamazine (DEC) on adult Wuchereria bancrofti, 31 infected men were randomly assigned to receive an initial single DEC dose of 1 mg/kg (n = 7), 6 mg/kg (n = 10), or 12 mg/kg (n = 14). Beginning 7 d later, the dosage of DEC and duration of treatment were progressively increased for 7-10 weeks. Physical examinations were performed to detect scrotal nodules and the scrotal area was examined by ultrasound (7.5 MHz transducer) to monitor the 'filaria dance sign' (FDS), the characteristic pattern of adult worm movement. Of 53 adult worm 'nests' that were detected by ultrasound, 22 (41.5%) were DEC-sensitive (FDS became non-detectable and a nodule became palpable at the site); 20 (37.7%) were not sensitive (FDS remained unchanged and detectable and no nodule developed), and 11 (20.8%) showed mixed responses (FDS remained detectable but a palpable nodule developed). All but one sensitive or mixed response occurred within 1 week after the initial single dose. Of 39 'nests' in men who initially received a single 6 or 12 mg/kg dose of DEC, 20 (51.3%) had sensitive responses compared to 2 (14.3%) of 14 'nests' in men who received a single 1 mg/kg dose (P = 0.04). Above 6 mg/kg, the macrofilaricidal effect of DEC did not increase with dose; a significant proportion of adult W. bancrofti were not susceptible to DEC during the study period.

To determine the extent to which Wuchereria bancrofti infections can be detected in asymptomatic amicrofilaraemic men in Greater Recife, Brazil, we studied 100 asymptomatic men who were long-term residents (> or = 15 years) of this filariasis-endemic area and who were amicrofilaraemic in 60 microL of capillary blood collected at night. Increasing amounts (1, 5, and 10 mL) of venous blood were collected in consecutive weeks, filtered, and examined for microfilariae; 27 men were eventually found to be microfilariaemic and 10 remained amicrofilaraemic but were found to be carriers of living adult W. bancrofti by ultrasound examination of the scrotal area. Thus, 37% of 'amicrofilaraemic' men (in 60 microL of blood) were found to be infected by more thorough investigation. Ultrasound is a valuable tool to identify adult worm infections in amicrofilaraemic persons, particularly for evaluation of serological assays and immunological studies in which the distinction between 'amicrofilaraemic adult worm carriers' and 'endemic normal subjects' is critical.

The ability of diethylcarbamazine (DEC) to kill adult Wuchereria bancrofti worms was evaluated by examining lymphatic nodules formed after treatment with 4 different treatment schedules of 193 males living in the endemic area of Greater Recife, Brazil. Lymphatic nodules appeared in the spermatic cord or upper extremities in 43 of 138 microfilaraemic individuals, in 3 of 30 amicrofilaraemic patients with filarial disease manifestations, and in 1 of 25 asymptomatic amicrofilaraemic residents of the endemic area treated with DEC. Fourteen of these nodules were surgically removed 10-150 d after the start of treatment. Regardless of the DEC dosage and schedule used, all nodules contained damaged and degenerating adult worms. An exuberant granulomatous process with large numbers of eosinophils and progressive fibrosis gradually developed around the dead parasites. The mechanism(s) by which DEC killed adult W. bancrofti could not be determined.

To determine the frequency with which living adult Wuchereria bancrofti can be detected by ultrasound in the scrotal area of men with filarial infection, we used a 7.5 MHz transducer to perform weekly ultrasound examinations on 100 microfilaraemic men (18-34 years old) from Greater Recife, Brazil. The peculiar pattern of movement that characterizes the adult worm image on ultrasound (the filaria dance sign) was detected in the lymphatic vessels of the spermatic cord in 80 men (bilaterally in 29 men). Among 20 men with no filaria dance sign, the geometric mean microfilarial density was 68/mL, compared with 238/mL and 775/mL among those with unilateral and bilateral filaria dance signs, respectively (P = 0.0001). The lymphatic vessels of the spermatic cord appear to be a common, and perhaps the principal, site of adult W. bancrofti in men with asymptomatic microfilaraemia. Studies are needed to define the relationship between the presence of filarial worms in the scrotal area and the development of filaria-associated morbidity.

Although the tropical pulmonary eosinophilia (TPE) syndrome of filarial aetiology has very distinctive clinical and immunological features, its clinical profile is not unique; other helminths sometimes induce similar presentations. We carefully evaluated 7 individuals with non-filarial TPE-like syndromes and found that serological tests based on detection of 'antifilarial' immunoglobulin (Ig) G, IgG4, and IgE antibodies that are usually considered diagnostic for filarial TPE were equally elevated in patients with non-filarial, TPE-like syndromes and were therefore unhelpful diagnostically. The apparent reasons were immunological hyper-responsiveness of such individuals and the shared (i.e., cross-reactive) antigenicity found in the filarial antigen preparations used routinely for diagnosis. Because appropriate treatment for those different pulmonary eosinophilia conditions requires different drugs and management, and because delay in effective treatment results in significant morbidity in such patients, diagnostic capabilities must be improved by identifying and obtaining unique antigens that can serologically discriminate between filarial TPE and other similar, but non-filarial, pulmonary eosinophilia syndromes.

Although the potent microfilaricidal activity of ivermectin is well established, its efficacy against adult Wuchereria bancrofti is unknown. We used longitudinal ultrasound examinations for periods of 3-9 months to assess directly the macrofilaricidal effect of a single 400 micrograms/kg dose of ivermectin in 15 men from Recife, Brazil who were infected with W. bancrofti. Before treatment, microfilarial densities ranged from 3 to 3098 microfilariae per mL of blood, and movements characteristic of the living adult worm (the 'filaria dance sign') were observed by ultrasound examination of the scrotal lymphatic vessels in all 15 men. Following treatment, microfilarial density was markedly reduced in all men, but the filaria dance sign remained unchanged in both location and pattern. Eight months after treatment, a dilated lymphatic vessel was surgically removed from one patient at the site of the filaria dance sign, and 3 intact adult worms were released. When given as a single 400 micrograms/kg dose, ivermectin had no observable effect on adult W. bancrofti. Therefore, prolonged suppression of microfilaraemia following treatment with ivermectin cannot be explained by a macrofilaricidal effect of the drug. Ultrasound is a valuable new tool for directly and rapidly assessing the macrofilaricidal efficacy of antifilarial drugs in lymphatic filariasis.

To assess the efficacy of diethylcarbamazine (DEC) in clearing Wuchereria bancrofti microfilariae (mf) from the circulation, we conducted a single blind hospital-based therapeutic trial of 3 DEC regimens. All patients were assessed by filtration of 1 mL of venous blood taken before and 1, 3, 6 and 12 months after DEC administration. The efficacy of a 12 d course of 6 mg/kg DEC once daily was identical to that of a similar course with 2 mg/kg given 3 times daily, indicating that split-dose treatment does not improve mf clearance over single daily drug administration. Microfilarial densities in patients treated only once with 6 mg/kg DEC remained significantly higher at 1, 3, and 6 months after treatment. However, all 3 treatment regimens proved equally effective in controlling microfilaraemia after 12 months, when 41, 42 and 40% of patients in the 3 treatment groups were amicrofilaraemic. These results suggest that a single DEC dose of 6 mg/kg administered annually or biannually may be a suitable regimen to control bancroftian filariasis in Recife, Brazil.

The effectiveness of single oral doses of ivermectin (200 or 400 micrograms/kg) and diethylcarbamazine (DEC, 6 mg/kg), preceded 4 d earlier by either placebo or very small doses of these drugs, was compared, over a 2-year period, in a double-blind trial in 67 microfilaraemic Brazilian men with bancroftian filariasis. Regimens containing ivermectin alone decreased the number of microfilariae significantly faster and more effectively for the first month after treatment than regimens containing DEC alone, but the latter were significantly more effective throughout the second year after treatment (1.7-8.2% of pretreatment levels with DEC vs. 12.6-30.8% with ivermectin during that period); the higher ivermectin dose showed a tendency towards more effectiveness than the lower dose. Most effective was the combination of ivermectin (20 micrograms/kg) followed 4 d later by DEC (6 mg/kg), with reduction of microfilaraemia to 2.4% of pretreatment levels at 2 years. Adverse reactions were well tolerated with all regimens, the reactions being significantly more generalized (i.e., fever) following ivermectin and localized (i.e., scrotal inflammatory nodules around dying adult worms) following DEC. Further trials of single-dose combination therapy vs. single high doses of ivermectin or DEC should determine the ideal regimen for treatment and control of bancroftian filariasis.

To determine the tolerance to diethylcarbamazine (DEC) treatment of patients with Bancroftian filariasis, 193 individuals (138 asymptomatic microfilaraemic, 30 amicrofilaraemic diseased patients and 25 asymptomatic amicrofilaraemic endemic residents) were enrolled in a prospective trial with different dose schedules, in a hospital and outpatient clinic setting in Brazil. Systemic adverse reactions, localized adverse reactions, and side effects, related to microfilariae, adult worms and the drug itself, were evaluated. Systemic reactions occurred irrespective of the DEC dose and schedule in about 40% of the microfilariae carriers, but not in amicrofilaraemic diseased patients or residents; they usually consisted of microscopic haematuria, followed by fever and malaise. Localized reactions were manifested by the appearance of inflammatory reactions, mainly in the scrotal area. Nodules containing degenerating adult worms developed mainly in the scrotal lymphatics of microfilaraemic patients, diseased amicrofilaraemic patients, and residents. Drowsiness, which increased with higher doses of DEC, was the most common side effect in both microfilaraemic and amicrofilaraemic individuals, followed by nausea and gastric upset. The results indicate that the occurrence of systemic and local adverse reactions was unrelated to either the dose of DEC or the pretreatment microfilarial density. The severity of systemic reactions was proportional to the microfilarial density. Side effects were dependent on the drug dosage irrespective of infection status.