Seroepidemiology of Strongyloides spp. Infection in Balimo, Western Province, Papua New Guinea

ABSTRACT.

Strongyloidiasis in Papua New Guinea (PNG) is poorly understood. There have been limited surveys describing the levels of endemicity in some regions of PNG, but in the Western Province, its occurrence and level of burden are unknown. This study aimed to determine the seroepidemiology of Strongyloides spp. seropositivity within a community located in the Balimo region of the Western Province. Plasma samples were collected from 120 adult participants and were subjected to anti-IgG Strongyloides spp. serological testing. Logistical regression analyses were performed to identify relationships between strongyloidiasis and attributes of sociodemography. In this cross-sectional cohort study, 22.5% (27/120; 95% CI: 15.9–30.8%) of participants were seropositive for strongyloidiasis. Participants with higher body mass indices were less likely to be seropositive for Strongyloides spp. infection (odds ratio [OR] = 0.85, P value = 0.008), and in the multivariable analysis, increasing units of age (adjusted OR [aOR] = 0.93, P value = 0.048) and participants ≤ 40 years old were associated with a decreased likelihood of Strongyloides spp. seropositivity (aOR = 0.07, P value = 0.034). The results from this study indicate that the occurrence of strongyloidiasis is high in the Western Province, PNG, and age is a determining factor of seroreactivity. This study provides evidence of endemic strongyloidiasis in this community and raises questions as to the impact of this neglected disease and other intestinal parasites on disease burden and comorbidities.

INTRODUCTION

Strongyloidiasis is a helminthic disease caused by three Strongyloides species known to parasitize humans: Strongyloides stercoralis and Strongyloides fuelleborni (fuelleborni) and Strongyloides fuelleborni (kellyi).¹ It is estimated that Strongyloides spp. infect more than 600 million people globally and are endemic in tropical and temperate climates.² Infection is acquired through skin penetration from infective filariform larvae that have contaminated the environment through infected feces. Therefore, transmission is maintained where sanitation and hygiene practices and infrastructure are inadequate.¹ In countries such as Papua New Guinea (PNG), the prevalence of the disease is considered high.^3,4 However, there are no recent reports on current disease occurrence, particularly in isolated rural communities in the Western Province.

Although S. stercoralis has a worldwide distribution,² the extent of its distribution and related pathologies remains primarily neglected and under-recognized.⁵ Clinical presentations of strongyloidiasis infection can range from acute and asymptomatic to chronic and symptomatic.⁶ Impaired function of the host’s immune system can result in uncontrolled parasite replication (hyperinfection), leading to visceral dissemination, which can have fatal consequences.^1,6,7 Strongyloides fuelleborni (kellyi) has been exclusively reported to parasitize humans in PNG.³ Since the discovery of S. f. (kellyi) in 1973, much of its distribution, clinical consequences, and reservoir of infection is unexplored.^8,9 From previous studies, S. f. (kellyi) has been described as an infection of infancy and childhood (< 15 years old), with the prevalence decreasing into adulthood.^3,10 Historical survey data have demonstrated that both S. stercoralis and S. f. (kellyi) have various levels of endemicity in multiple provinces.^11–15 Previous large-scale survey data of the disease were collected decades ago and consisted of only fecal (stool) examination results collected from young children, which has been demonstrated to be an insensitive indicator of disease.¹⁶

In addition to causing direct pathology in the gastrointestinal tract, infections with soil-transmitted helminths (STHs) have also been implicated as impacting the clinical outcomes of other infectious diseases, including HIV, malaria, and tuberculosis (TB).¹⁷ In TB-helminth co-endemic communities, the presence of STHs impacts the protective TB immunity of individuals^18,19 and can alter disease manifestations,^20,21 which may lead to complications in patient management.^22–24

In PNG, the distribution of preventive chemotherapy to school-aged children is the mainstay of parasite control,²⁵ with ongoing efforts to eliminate malaria and filariasis.^25–27 Strategies for management of STHs are less common, with fewer than 75% of school-aged children receiving preventive chemotherapy, and do not include treatment of Strongyloides spp. infections.²⁸ The purpose of this study was to investigate the seroepidemiology of strongyloidiasis in rural PNG and to investigate population determinants of Strongyloides sp. seropositivity.

MATERIALS AND METHODS

Study design, setting, cohort characteristics, and ethics.

This cross-sectional study involved participants recruited into a broader program investigating TB epidemiology in the Balimo region of the Western Province, in collaboration with the Balimo District Hospital (BDH).²⁹ Community members over the age of 16 years were eligible for participation, including patients from the BDH. Demographic information and cultural characteristics of the population were obtained via a structured questionnaire. Anthropometric measurements such as height and weight were also noted to calculate crude body mass index (BMI). Ethics approval was obtained from the James Cook University (JCU) Human Research Ethics Committee (H6432 and H8015) and the PNG Medical Research Advisory Committee (MRAC 19.21). All participants provided either written and/or verbal informed consent.

Sample collection and strongyloidiasis serodiagnosis.

In conjunction with TB investigations, blood was drawn from participants into four separate heparin QuantiFERON-TB Gold plus (QFT-G+) tubes (Qiagen Inc, Germantown, MD). Resultant plasma from the “null” tube was used for strongyloidiasis serology. Previous work from our group (data not shown) determined that antibody integrity is maintained throughout the sample processing and storage procedures required for the QFT-G+ assay. Plasma samples of 120 participants were used to determine Strongyloides spp. seroreactivity. A commercial In Vitro Diagnostics Strongyloides Serum Antibody Detection Microwell ELISA testing kit from DRG Instruments GmbH (Marburg, Germany; sensitivity 92.3% and specificity 97.4%)³⁰ was used per the manufacturer’s instructions. This assay detects anti-IgG Strongyloides spp. antibodies using an ELISA plate coated with S. stercoralis somatic antigens. In this study, an absorbance reading of ≥ 0.4 optical density (OD) units indicated a positive result; OD units between 0.2 and 0.4 were deemed intermediate, whereas an OD unit ≤ 0.19 was considered negative. Cutoff ranges were adopted from laboratory standards in Western Australia.³¹ Intermediate results were grouped with negative results to avoid overestimating the prevalence of strongyloidiasis.

Stool collection and other parasitological examinations.

Stool samples were obtained from participants and processed and stored in sodium acetate formalin within 6 hours. Samples were stored at room temperature in PNG and then at 4°C after being delivered to JCU, Townsville, Australia. The identification of STHs was determined by microscopy through the observation of ova or larvae on standard wet preparations, under ×100 magnification. Two to three wet preparations were examined per sample.

Data analysis.

Statistical analyses were performed using GraphPad prism 9 (Prism for Windows, version 9.2.0.332; GraphPad Software, San Diego, CA). Participant plasma samples analyzed were matched with questionnaire responses and other parasitological data. Four participants were missing data on BMI and one on age. Further data regarding mother’s and father’s clans from Siboko and Gasinapa, respectively, were omitted from the analysis because of the small sample size, as were participants who answered “unknown” on the questionnaire. Complete case analysis was used. Descriptive summaries are presented as frequencies and percentages for categorical variables. A logistics regression model assessed crude associations between predictors and the outcome (Strongyloides spp. status). Variables considered in the multivariable model were preselected based on their clinical importance (age, sex, BMI, and helminth infection) and other variables with P values ≤ 0.25. A multivariable logistic regression analysis was conducted iteratively to assess the association of exposure variables with the outcome. Adjusted odds ratio (aOR) estimates and 95% CIs are presented. Inference was based on a 5% level of significance.

RESULTS

General characteristics of participants with a Strongyloides spp. seropositive reaction.

Results are presented in Table 1. Overall, Strongyloides spp. seropositivity was 22.5% (27/120; 95% CI: 15.9–30.8%). Those aged ≤ 40 years had a higher rate of seropositivity (28.9%; 95% CI: 17.0–44.8%) than those ≥ 41 years (19.8%; 95% CI: 12.5–29.7%) but were also under-represented in this study (38/119, 31.9%; 95% CI: 24.2–40.8%). Although females were overrepresented at 67.5% (81/120; 95% CI: 58.7–75.2%) in this cohort compared with the general population,³² males had a greater seropositivity rate (28.2%; 95% CI: 16.5–43.8%) than females (19.8%; 95% CI: 12.5–29.7%). Of participants seropositive for strongyloidiasis, a majority (57.1%; 95% CI: 25.0–84.2%) were underweight. Additionally, more seropositive participants (25.0%; 95% CI: 17.0–35.2%) resided outside Balimo compared to those from within the Balimo region (16.7%; 95% CI: 7.9–31.9%).

Table 1.

Sociodemographic characteristics and parasitological data of individuals seropositive for Strongyloides spp. infection

Factor	Group	Number analyzed	Number seropositive	Occurrence (%)	95% CI
All participants	NA	120	27	22.5	15.9–30.8
Age	≤ 40 years	38	11	28.9	17.0–44.8
	≥ 41 years	81	16	19.8	12.5–29.7
Sex	Male	39	11	28.2	16.5–43.8
	Female	81	16	19.8	12.5–29.7
Residency (location)	Balimo (central)	36	6	16.7	7.9–31.9
	Not Balimo (away from central)	84	21	25.0	17.0–35.2
BMI categories	Normal (18.6–24.9 kg/m2)	62	16	25.8	16.6–37.9
	Underweight (≤ 18.5 kg/m2)	7	4	57.1	25.0–84.2
	Overweight/obese (≥ 25 kg/m2)	47	7	14.9	7.4–27.7
Other STHs
Total positive for any STH		50	14	28.0	17.5–41.7
STH coinfection		41	8	19.5	10.2–34.0
Poly-STH infection		9	4	44.4	18.9–73.3

BMI = body mass index; NA = not applicable; STH = soil-transmitted helminth.

Other STHs and Strongyloides spp. seroreactivity.

In this cohort, 41.7% (95% CI: 33.5–50.6%) of participants were infected with at least one other type of STH excluding Strongyloides spp. seropositive individuals. The total proportion of seropositive individuals with an STH coinfection was 28% (95% CI: 17.5–41.7%). A small portion of participants, 7.5% (95% CI: 4.0–13.6%), had polyparasitic infection with other STHs, with 44% (95% CI: 18.9–73.3%) also seropositive for strongyloidiasis.

Determinants of Strongyloides seropositivity.

Results of the univariate analysis are presented in Table 2. We found a 15% decrease in the odds of Strongyloides spp. seropositivity for a unit increase in BMI (OR = 0.85; 95% CI: 0.76–0.95; P value = 0.008). There was insufficient evidence of an association between age, sex, location, BMI, and others with Strongyloides spp. seropositivity. Results of the multivariable analysis, adjusted for confounders, are presented in Table 3. Evidence suggests a 7% decrease in the odds of Strongyloides spp. seropositivity for a unit increase in age (aOR = 0.93; 95% CI: 0.85–1.0, P value = 0.048). It was also found in the multivariable analysis that compared with those aged ≥ 41 years, aOR for those ≤ 40 years was 0.07 (95% CI: 0.005–0.73, P value = 0.034) for Strongyloides spp. seropositivity. This finding contrasts with the unadjusted OR values found for this age group in the univariate analysis (Table 2), which indicated an OR of 1.66 (95% CI: 0.67–4.01, P value = 0.268) for strongyloidiasis. There were no other statistically significant associations with Strongyloides spp. serology outcome.

Table 2.

Univariate analysis of sociodemographic, cultural, and parasitological determinants for Strongyloides spp. seropositivity

Determinant	Factors		Univariate
		Number analyzed	OR (95% CI)	Standard error	P value
Age	≥ 41 years	81	Ref.	Ref.	Ref.
	≤ 40 years	38	1.66 (0.67–4.01)	0.454	0.268
Age		119	0.97 (0.25–1.00)	0.016	0.071
Sex	Female	81	Ref.	Ref.	Ref.
	Male	39	1.6 (0.65–3.86)	0.452	0.301
Residency (location)	Balimo (central)	36	Ref.	Ref.	Ref.
	Not Balimo (away from central)	84	1.67 (0.64–4.92)	0.513	0.320
Education level	Secondary	61	Ref.	Ref.	Ref.
	No/primary	59	1.39 (0.59–3.35)	0.440	0.452
Employment status	Unemployed	81	Ref.	Ref.	Ref.
	Employed	39	1.05 (0.41–2.57)	0.465	0.916
BMI categories	Normal (18.6–24.9 kg/m2)	62	Ref.	Ref.	Ref.
	Underweight (≤ 18.5 kg/m2)	7	3.83 (0.77–21.28)	0.817	0.100
	Overweight/obese (≥ 25 kg/m2)	47	0.50 (0.18–1.31)	0.502	0.171
BMI		116	0.85 (0.76–0.95)	0.051	0.008*
Father’s clan	Awala	18	Ref.	Ref.	Ref.
	Asipali	17	0.56 (0.10–2.74)	0.826	0.479
	Gasinapa	6	0.52 (0.02–4.45)	1.215	0.591
	Lalamana	35	0.90 (0.25–3.43)	0.653	0.872
	Siboko	3	Nd†	Nd†	Nd†
	Tabama	7	1.04 (0.12–6.87)	0.988	0.968
	Wabadala	15	0.40 (0.05–2.24)	0.924	0.321
	Wagumisi	14	0.43 (0.05–2.44)	0.928	0.367
Mother’s clan	Awala	17	Ref.	Ref.	Ref.
	Asipali	26	0.44 (0.092–1.94)	0.761	0.276
	Gasinapa	3	Nd†	Nd†	Nd†
	Lalamana	10	0.27 (0.01–2.05)	1.181	0.263
	Siboko	11	0.240 (0.01–1.82)	1.176	0.225
	Tabama	5	0.60 (0.03–5.50)	1.238	0.680
	Wabadala	23	1.54 (0.41–6.22)	0.683	0.525
	Wagumisi	15	0.37 (0.05–2.08)	0.532	0.283
Positive for other STH	No	70	Ref.	Ref.	Ref.
	Yes	50	1.71 (0.72–4.08)	0.440	0.225
STH co-infection	No	79	Ref.	Ref.	Ref.
	Yes	41	1.18 (0.47–2.84)	0.445	0.721
Poly-STH infection	No	111	Ref.	Ref.	Ref.
	Yes	9	3.06 (0.71–12.48)	0.711	0.115

BMI = body mass index; Nd = not done; OR, odds ratio; Ref. = reference category; STH = soil-transmitted helminth.

^*Statistically significant (P ≤ 0.05).

^†Sample size too small.

Table 3.

Multivariable analysis of sociodemographic, cultural, and parasitological determinants for Strongyloides spp. seropositivity

Determinant	Factors		Multivariable
		Number analyzed	aOR (95% CI)	Standard error	P value
Age	≥ 41 years	81	Ref.	Ref.	Ref.
	≤ 40 years	38	0.07 (0.005–0.73)	1.255	0.034*
Age		119	0.93 (0.85–1.0)	0.039	0.048*
Sex	Female	81	Ref.	Ref.	Ref.
	Male	39	3.00 (0.88–10.94)	0.634	0.083
BMI categories	Normal (18.6–24.9 kg/m2)	62	Ref.	Ref.	Ref.
	Underweight (≤ 18.5 kg/m2)	7	1.59 (0.10–25.48)	1.402	0.742
	Overweight/obese (≥ 25 kg/m2)	47	1.69 (0.25–12.18)	0.975	0.593
BMI		116	0.86 (0.64–1.11)	0.138	0.277
Mother’s clan	Awala	17	Ref.	Ref.	Ref.
	Asipali	26	0.54 (0.10–2.70)	0.851	0.455
	Gasinapa	3	Nd†	Nd†	Nd†
	Lalamana	10	0.56 (0.024–5.42)	1.271	0.653
	Siboko	11	0.35 (0.01–3.21)	1.266	0.402
	Tabama	5	1.82 (0.047–14.09)	1.345	0.901
	Wabadala	23	2.29 (0.51–11.22)	0.776	0.285
	Wagumisi	15	0.69 (0.77–4.78)	1.011	0.714
Positive for other STH	No	70	Ref.	Ref.	Ref.
	Yes	50	1.38 (0.41–4.54)	0.609	0.599
Poly-STH infection	No	111	Ref.	Ref.	Ref.
	Yes	9	2.65 (0.40–18.86)	0.963	0.312

aOR = adjust odds ratio; BMI = body mass index; Nd = not done; Ref. = reference category; STH = soil-transmitted helminth.

^*Statistically significant (P ≤ 0.05).

^†Sample size too small.

DISCUSSION

This cross-sectional study aimed to provide evidence on the occurrence of strongyloidiasis within a rural community in PNG and to indicate population determinants of Strongyloides spp. seroreactivity. In this study, 22.5% of participants were seropositive for strongyloidiasis in the Balimo region of the Western Province, which may be considered high; however, the prevalence rates reported here are comparable to rates in neighboring countries.^2,33 Strongyloidiasis has been described in previous studies in PNG, highlighting disease endemicity within certain regions³⁴ and demonstrating a scattered distribution across different geographical areas.³⁵ For example, studies conducted in the 1980s within the six localities in the Purari-Kikori delta area identified that Strongyloides spp. prevalence was rare (1%),¹³ whereas infection rates diagnosed by traditional parasitological tools in other isolated communities in the Kerema area, Gulf Province, were similar to the findings presented in our study (25%).³⁵

Our study found that participants younger than 40 years had a higher rate of strongyloidiasis seropositivity than participants older than 41 years. However, multivariable analysis adjusted for sex, age, BMI, and mother’s clan suggests that those aged up to 40 years had 93% decreased odds of strongyloidiasis compared with those aged 41 plus years. Previous surveys in PNG have reported a decreasing trend of strongyloidiasis and intensity of infection with increasing age, particularly for S. f. (kellyi).^10,11,15 This observation of an age-related decrease in prevalence rate is inconsistent with S. stercoralis infections, as this parasite can be maintained chronically within the host by “autoinfection.”^36–38 In previous studies in PNG, young children were found to be heavily burdened by Strongyloides spp., with infection rates approaching 80% in children < 5 years old, with rates decreasing to < 20% in adults (> 15 years old).^3,10,12 It is not clear whether the anti-Strongyloides spp. IgG assay utilized in this study can discriminate exposure/infection among other coexisting Strongyloides species.

In our study, the seroprevalence rate was greater in males than in females, but with associations lacking statistical significance. This trend has been reported in other neighboring countries.^36,38–40 This may reflect differences in daily activities, behavior, and interactions with the environment⁴¹ and that females, particularly those of reproductive age, are more likely to be targeted for health interventions, promoting an inherent sex bias in health survey data.^42,43

Food insecurity, changing diets in urban communities, and poor access to health services are a few adversities significantly influencing health in PNG communities.^44,45 An inverse relationship between seropositivity and increasing BMI was found in this study. Although this relationship lacked statistical significance in the multivariable analysis, it does imply that those classified as overweight (≥ 25 kg/m²) are less likely to be exposed or infected with Strongyloides spp. Societal and nutritional transitions in PNG, from rural subsistence lifestyles to peri-urban occupations and westernized, store-bought foods may reduce an individual’s risk of acquiring communicable diseases from the environment. This reduced risk of exposure/infection may be partly encouraged through a decrease in environmental interactions because of altered, urbanized behaviors.⁴⁶ Conversely, previous studies on children from PNG have shown that the intensity of Strongyloides spp. burden bears an inverse relationship with nutritional status; however, results were considered inconclusive in both studies because of small sample sizes.^15,47 Other studies describing the impact of strongyloidiasis on pregnant women and children found that infection was associated with low birth weight, wasting, and stunting,^48,49 and another found no overall significant relationship, comparable to findings presented here.⁴⁰ This study cannot determine the temporal causation for nutritional status, but the results suggest a complex relationship dynamic between disease acquisition, nutrition status, and interactions with the environment. Longitudinal studies or crossover, randomized, controlled trials could expand on this finding.

We found a trend for strongyloidiasis seropositivity associated with residence villages outside the main Balimo township. Although our result lacks statistical significance, it is consistent with previous studies in PNG¹⁵ and other countries.^36,37,49

Interestingly, although not statistically significant, mother’s but not father’s clan may be related to Strongyloides spp. seropositivity. Social-cultural factors can influence disease states and are an under-recognized contributor.⁵⁰ Various factors could contribute to this trend, as different clans may have different cultural practices in daily activities and may reside at certain localities that may influence the risk of exposure.

Co-endemicity of STHs has been frequently identified in PNG. A thorough study identifying other parasites within this community is currently underway. However, preliminary data reveal that approximately 42% of study participants were infected with other STHs, detected initially by stool microscopy. The results from this study suggest that seropositivity could be associated with other general helminth infections and poly-helminth coinfections; however, in the adjusted model, other STHs were not predictor variables of Strongyloides spp. seroreactivity outcome. The overlapping occurrence of STHs with strongyloidiasis in this community highlights the shared transmission dynamics of helminths. Typically, these helminths are acquired via the fecal-oral route by ingesting infective larvae or embryonated eggs from contaminated food and water sources.^51,52 However, skin penetration by infective filiform larvae from contaminated soil is unique to hookworm and Strongyloides species, highlighting their co-endemicity and clinical association.⁵³

Using serology as the basis of strongyloidiasis diagnosis is contentious.⁵⁴ Previous studies highlight that this method of diagnosis can exaggerate strongyloidiasis prevalence owing to cross-reactivity with other helminth infections. Furthermore, it is challenging to delineate recent infections from past (and treated) infections.⁵⁵ Although a few studies have demonstrated that after treatment of strongyloidiasis, antibody titers decrease over time, usually resulting in seronegative status.^54,56 ELISA OD cutoff values used in the current study were based on previous studies from endemic locations within Australia³¹ to ensure that only strong positive seroreactions were considered when determining prevalence rates, which may, to some extent, reduce false positives associated with STH cross-reaction. This higher cutoff value, however, increases the risk of false-negative test results. Strongyloides spp. IgG antibodies typically appear 6 weeks after initial exposure and remain elevated during chronic infections.⁵⁷ Although beyond the scope of the current study, additional samples may have captured more IgG-positive cases in this community. Antibody cross-reactivity between filaria and Strongyloides spp. antigens have also been described,^30,58 and Wuchereria bancrofti, a causative agent of filariasis, is endemic in parts of PNG, although its distribution is uneven.²⁷ Studies are underway to determine the prevalence and influence of these and other parasites in this community. Regardless, enhancing the reliability of strongyloidiasis diagnosis requires simultaneous use of other parasitological tools.⁵⁹ However, in the absence of these tools in rural settings, the utility of serology for population screening for strongyloidiasis is recommended.⁶⁰

Health-related behavioral and clinical data from large-scale surveys in parasite endemic areas are recommended for future studies to enable understanding of the occurrence of and determinant factors for strongyloidiasis and other STHs in PNG. Targeted and culturally grounded approaches to public health policies and interventions based on rigorous epidemiological studies enhance their sustainability within communities.⁶¹

CONCLUSION

Strongyloidiasis has a high prevalence in the Balimo region of the Western Province, PNG. This is the first study describing the seroepidemiology of Strongyloides spp. and sociodemographic associations of seropositivity in this region. Age was considered a determinant of Strongyloides spp. seropositivity in this study, and although other sociodemographic and parasitological characteristics showed trends toward associations with Strongyloides spp. seropositivity, none were statistically significant. These relationships, however, do provide a direction for future investigations. The challenges of diagnosing strongyloidiasis in resource-limited settings and identifying clinical manifestations of the disease highlight the importance of considering the differential diagnosis of intestinal and other associated diseases in rural PNG communities.