Strongyloides stercoralis Infection at Different Altitudes of the Cusco Region in Peru

Abstract.

Strongyloides stercoralis affects 30–100 million people worldwide. The burden is underestimated because of the paucity of studies, limited geographical areas surveyed, and poor quality of diagnostic tests. This study aimed at determining the epidemiology of strongyloidiasis using sensitive microscopy testing in rural populations living at different altitudes in Cusco, Peru. Data were collected from subjects aged > 3 years living in Quellouno (elevation 2,600 ft) and Limatambo (elevation 8,379 ft) districts. Subjects provided one fresh stool sample and answer a standardized questionnaire. Fresh stool was tested on site using the Baermann’s test and agar plate culture. Formalin-preserved stool was tested by rapid sedimentation. Eighty percent (585/715) of eligible subjects consented to participate; after excluding subjects with missing data, 65% (462/715) were included. Fifty-five percentage were female; the median age was 33 years (interquartile range 13–52), and 72% had government health insurance. Half had intestinal parasites, and Strongyloides was the most common (24.5%) followed by Giardia (15.5%), Blastocystis (14.9%), and hookworm (11.5%). The agar plate culture detected more cases of Strongyloides than Baermann’s or sedimentation tests. Strongyloides infection was more common at low altitude (26.4%) than at high altitude (18.6%), but the difference was not statistically significant (P = 0.08). Older age, walking barefoot, bathing in rivers/streams, and using municipal sewage were associated with strongyloidiasis. Strongyloides was the most prevalent parasite in the areas studied and was associated with demographic, socioeconomic, and sanitary factors.

INTRODUCTION

Strongyloidiasis, caused by the nematode Strongyloides stercoralis, is estimated to affect 30–100 million people worldwide.¹ However, based on burden estimates for other soil-transmitted helminths, specifically hookworm, Bisoffi et al.² argue that a more accurate number of people infected with Strongyloides is 370 million. Although Strongyloides is a cosmopolitan parasite, the infection is more prevalent in tropical and subtropical climates. In addition, the prevalence of infection within highly endemic countries is likely to vary significantly according to the environmental conditions. In most situations, only limited areas of each country are surveyed for strongyloidiasis and a limited number of studies per country are available, thus preventing a clear understanding of the epidemiology of the infection.³

Female Strongyloides parasites live within the epithelium of the small intestine where they produce eggs that hatch and release rhabditiform larvae into the intestinal lumen. These larvae are shed in the feces; however, they may mature into infective filariform larvae in the intestine to cause autoinfection.⁴ Autoinfection perpetuates strongyloidiasis in the host, which can remain undiagnosed for many years.² In people who become immunosuppressed, the parasite is able to invade multiple organs, causing life-threatening disseminated disease with a case fatality rate of up to 90%.⁴ Transmission of strongyloidiasis requires specific soil conditions (i.e., moisture, temperature, and acidity) to allow the free-living filariform larvae to develop.⁵ Host behaviors that allow direct contact of the skin with the contaminated soil favor transmission.⁶ Few studies compare the prevalence of strongyloidiasis among populations with different behaviors and environmental conditions.

The diagnosis of strongyloidiasis often relies on microscopic examination of stool specimens. The methods used for detection of Strongyloides larvae include direct stool microscopy, agar plate culture, and multiple concentration techniques, including sedimentation, flotation, and the Baermann’s test.^7,8 The sensitivity of each method varies significantly, with the agar plate culture and Baermann’s tests being the most sensitive.⁹ Serologic tests for Strongyloides IgG using recombinant antigens NIE and Strongyloides stercoralis immunoreactive antigen have demonstrated high sensitivity and specificity, but the availability and performance of commercial versions of these tests are still a problem in endemic areas, limiting their use.^8,10–13 Several studies have evaluated polymerase chain reaction tests for the diagnosis of Strongyloides with inconsistent results and a large variation in sensitivity.^7,14,15 Thus, the low sensitivity of the diagnostic methods used in epidemiologic studies hinders the evaluation of the burden and distribution of this infection in humans.

Peru has a high prevalence of Strongyloides infection (between 10% and 20%).¹⁶ Studies in Peru have shown significant variations in prevalence, ranging from < 1% to 40% depending on the area studied and the diagnostic methods used.¹⁷ Recent reviews on the prevalence of Strongyloides in Latin America and Peru suggested a very low prevalence in the highlands of Andes Mountains (prevalence < 1.5%).^16,17 The heterogeneity of the studies and diversity of diagnostic methods used limited the understanding of strongyloidiasis epidemiology at high altitude in South America. The objective of the present study was to determine the epidemiology of strongyloidiasis using a combination of sensitive microscopy tests among two populations living at different altitudes in Cusco, Peru.

METHODS

A secondary analysis of data primarily collected for another study evaluating a Strongyloides diagnostic method was performed to evaluate the prevalence and factors associated with S. stercoralis infection at different elevations. The primary cross-sectional study was conducted in the Quellouno (elevation 2,600 ft) and Limatambo (elevation 8,379 ft) districts of the Cusco region in Peru (Figure 1). The Quellouno district in the La Convencion province has a total population of 18,089 and is located in the eastern slope of the Andes Mountains, referred to as the high jungle, with an average yearly temperature of 26°C and a rainy season from September to April averaging 36 mm of monthly rainfall. The Limatambo district in the Anta province has a population of 9,801 and is located in an inter-Andean valley with an average yearly temperature of 13°C and a rainy season from November to March averaging 137 mm of monthly rainfall. The human development index for Quellouno and Limatambo was 0.2931 and 0.2947 in 2012, respectively. Most of the population is composed of subsistence farmers living in adobe houses with dirt floors. Three communities in Quellouno (Putucusi, Alto Putucusi, and San Martin) and one community in Limatambo (Sauceda) were selected based on convenience (i.e., accessibility) for data collection between August 2015 and June 2016.

Figure 1.

Map of the Cusco region showing the geographic relationship between the study sites. The inset shows the map for the south part of Peru (adapted from www.weatherspark.com Cedar Lake Ventures, Inc.).

The study was carried out in collaboration with the local ministry of health and community authorities who were responsible for informing the population about the study. Male and female subjects aged 3 years or older residing in the selected communities were eligible to participate. Subjects were enrolled at their homes after providing informed consent. Children aged less than 18 years were required to provide assent in addition to the parent’s informed consent before enrollment. Bilingual (Spanish and Quechua) field workers conducted individual interviews with the participants and recorded their responses in standardized questionnaires. Information about demographics, socioeconomic status, and potential risk factors for Strongyloides infection was collected from participants. They were provided stool collection supplies and instructions to collect one fresh specimen in the morning of the scheduled household visit. Freshly produced specimens were collected and transported immediately to an onsite laboratory mounted for this study at the local health centers and staffed by experienced laboratory technicians. Aliquots from each stool sample were immediately tested by the Baermann’s test and agar plate culture to detect S. stercoralis larvae as previously described.¹⁸ Aliquots of each stool sample were preserved in 10% formalin to perform the Lumbreras rapid sedimentation test to detect helminth eggs at the laboratory of the Universidad Peruana Cayetano Heredia and University of Texas Medical Branch Collaborative Research Center in Cusco city.¹⁹

The data were entered and analyzed using the Statistical Package for the Social Sciences version 18.0 (SPSS Inc., Chicago, IL). Mean (±SD), medians with interquartile range (IQR), and frequencies were calculated to describe the distribution of the variables. The chi-squared test, Student’s t-test, and Mann–Whitney U-test were used to compare variables. Backward logistic regression was used to model Strongyloides and factors associated with the infection. A P < 0.05 was considered statistically significant.

The study protocol was approved by the Universidad Peruana Cayetano Heredia Institutional Review Board.

RESULTS

The census of the population for the four communities revealed 715 subjects eligible to participate in the study, and 585 (81.8%) subjects signed an informed consent form. Of these, 123 were excluded from the analysis because of the lack of complete stool test results (i.e., insufficient sample) or epidemiological information. A total of 462 (64.6%) subjects were included in the data analysis: 74.5% (344/462) from the low-altitude communities (Alto Putucusi, Putucusi, and San Martin) and 25.5% (118/462) from the high altitude-community (Sauceda) (Table 1). The median age of the participants was 33 years (IQR 13–52), and 54.9% (254/462) were female. Most participants lived in houses with dirt floors (69.9%, 305/436) and tin roofs (94.5%, 411/435). However, almost all (98%, 431/440) had drinking water tubed into their homes for at least a few hours per day. The majority (72.4%, 317/438) had health insurance provided by the government.

Table 1.

Characteristics of the study population

		Frequency (n = 462)	%
Community	Alto Putucusi	106	22.9
	Putucusi	174	37.7
	San Martin	64	13.9
	Sauceda	118	25.5
Median age (IQR)	33 years (13–52)
Gender	Female	254	54.9
	Male	208	45.1
Median number of diarrhea episodes/year (IQR)	2 (0–3)
Floor material (n = 436)	Dirt	307	70.4
	Not dirt	129	29.6
Roof material (n = 437)	Tin	411	94.1
	Tile	16	3.7
	Concrete	8	1.8
Source of drinking water (n = 440)	Municipal tubed	431	98.0
	River	3	0.7
	Irrigation channel	3	0.7
Government health insurance (n = 438)	Yes	317	72.4
	No	121	27.6

IQR = interquartile range.

Feces were eliminated using the municipal sewage system (59.6%, 261/438), improved latrines (26.5%, 116/438), septic tanks (9.6%, 42/438), simple pits (3.7%, 16/438), and the open field (0.7%, 3/348). Most subjects reported inconsistent or no wearing shoes while defecating (80.7%, 356/441) and inconsistent or no hand washing after defecating (67.9%, 296/436). The most frequently wore footwear were sandals (69.8%, 308/441) and shoes (23.6%, 104/441). The majority reported not walking (44.5%, 190/427) or rarely (47.5%, 203/427) walking barefoot outside their home. Almost 60% (262/436) reported working in their farm often or every day. Almost all subjects performed personal hygiene in a shower or bathtub (98%, 433/442).

Thirty-nine percent (38.6%, 177/458) reported a past medical history; one-third of these reported malaria (29.4%, 52/177), 2.7% (12/177) anemia, and 1.8% (8/177) each typhoid, dyspepsia, and hepatitis. Almost half of these subjects (47.5%, 84/177) reported other diverse medical problems. Diarrhea in the previous 12 months was reported by 70.5% (324/459) of the subjects, and the median number of diarrheal episodes in that period was 2 (IQR 0–3). The most common symptoms reported by the participants in the 3 months before the study were cough, anorexia, and fatigue (Table 2).

Table 2.

Symptoms in the prior 3 months reported by the participants and their frequency according to the Strongyloides infection status

	Total		Strongyloides (+)		Strongyloides (−)
Symptoms	%	N	%	N	%	N	P-value
Cough	20.6	88/428	24.8	26/105	19.2	62/323	0.22
Anorexia	15	64/428	11.4	12/105	16.1	52/323	0.24
Fatigue	12.1	52/428	7.7	8/104	13.6	44/324	0.11
Bloating	11	47/429	4.8	5/105	13	42/324	0.02
Pallor	10.1	43/427	6.7	7/104	11.1	36/323	0.19
RUQ pain	10	43/428	10.6	11/104	9.9	32/324	0.83
Weight loss	8	34/427	6.7	7/104	8.4	27/323	0.59
Diarrhea	6.1	26/427	7.7	8/104	5.6	18/323	0.43
Rash	5.8	25/428	3.8	4/104	6.5	21/324	0.47
Jaundice	5.6	24/428	1	1/105	7.1	23/323	0.01
Nausea/vomiting	5.2	22/427	2.9	3/104	5.9	19/323	0.31

RUQ = right upper quadrant. N is different from total enrolled subjects because of no response to symptoms questions.

Half (50.2%, 232/462) of the participants had at least one intestinal helminth or protozoan detected by microscopy. Strongyloides stercoralis (24.5%, 113/462) was the most common intestinal helminth, and Giardia spp. (15.8%, 63/462) was the most common protozoan. Blastocystis hominis (14.9%, 69/462), hookworm (11.5%, 53/462), Trichuris trichiura (5.8%, 27/462), Hymenolepis nana (3.5%, 16/462), and Ascaris lumbricoides (1.9%, 9/462) were less commonly found. The agar plate culture test detected the most cases of Strongyloides (18.4%, 85/462), followed by the Lumbreras rapid sedimentation method (12.8%, 55/431), and the Baermann’s test (10.2%, 47/462). Of note, the agreement between the agar plate culture and the Lumbreras rapid sedimentation (Kappa = 0.36, P < 0.001) or the Baermann’s test (Kappa = 0.47, P < 0.001) was only moderate.

Except for bloating and jaundice, the symptoms reported by subjects with strongyloidiasis were not significantly different from the symptoms reported by subjects without the infection (Table 2). Strongyloides infection was significantly associated with older age, certain socioeconomic characteristics (house roof material, owning poultry, and having government health insurance), and some exposures, such as using the municipal sewage system, bathing in rivers or streams, and walking barefoot (Table 3). Strongyloides infection was more common among those living at lower altitude than those living a higher altitude (26.4% versus 18.6%) and among those working often in farms than those doing so infrequently or not at all (26.7% versus 21.8%), but the differences did not reach statistical significance. The backward logistic regression analysis showed that those who bathe in rivers and streams, used the municipal sewage system, walked barefoot often or always, and were older were significantly more likely to have Strongyloides infection. By contrast, those who raised chicken and had government-provided health insurance were less likely to have Strongyloides (Table 4).

Table 3.

Characteristics of the population according to the Strongyloides infection status

Characteristic		Strongyloides (+)		Strongyloides (−)
		n	%	n	%	P-value
Disposal of feces	Municipal sewage	79	69.9	182	52.1	0.001
	Other	34	30.1	167	47.9
Household roof	Tin	109	96.5	302	86.5	0.003
	Other	4	3.5	47	13.5
Raises chicken at home	Yes	81	71.7	292	83.7	0.005
	No	32	28.5	57	16.3
Age in years	Median (IQR)	36.5	(25–53.5)	32	(11.25–51)	0.01
Raises ducks at home	Yes	22	19.5	108	30.9	0.01
	No	91	80.5	241	69.1
Has government insurance	Yes	74	65.5	267	76.5	0.02
	No	39	34.5	82	23.5
Personal hygiene	Shower/bathtub	103	95.4	330	98.8	0.04
	River/stream	5	4.6	4	1.2
Walks around barefoot	Often/always	13	12.4	21	6.5	0.05
	Seldom/never	92	87.6	301	93.5
Elevation	Low	91	80.5	253	72.5	0.08
	High	22	19.5	96	27.5
Works in the farm	Often/always	70	64.8	192	58.5	0.24
	Seldom/never	38	33.2	136	41.5
Gender	Female	57	50.4	197	56.4	0.26
	Male	56	49.6	152	43.6

IQR = interquartile range.

Table 4.

Backward logistic regression analysis of variables associated with Strongyloides infection

Characteristic	Category	Adjusted OR	95% CI		P-value
Personal hygiene	River or stream	5.91	1.32	26.38	0.020
Disposal of feces	Municipal sewage	2.51	1.47	4.28	0.001
Walks around barefoot	Often/Always	2.23	1.02	4.9	0.043
Age	Older	1.01	1	1.02	0.031
Raises chicken	Yes	0.46	0.26	0.81	0.007
Government health insurance	Yes	0.49	0.29	0.83	0.008

OR = odds ratio.

DISCUSSION

Strongyloidiasis is an underestimated parasitic public health problem in developing countries. In this study, one of four subjects evaluated was infected with Strongyloides and half of the subjects diagnosed with a parasite had strongyloidiasis. Strongyloides infection was associated with older age; certain socioeconomic characteristics, such as having a tin roof, owning poultry, and having government health insurance; and exposures, such as using the municipal sewage system, bathing in rivers or streams, and walking barefoot.

Importantly, the prevalence of Strongyloides at high altitude was not statistically different than the prevalence at low altitude. This finding contrasts with several reports from Peru and highlights the importance of using Strongyloides-specific microscopy methods to obtain more accurate estimates of this parasite’s burden. Similarly, a recent study from Bolivia using the same microscopy methods (sedimentation, Baermann’s, and culture) showed a Strongyloides prevalence of 6.4% at an elevation 2,500 m that was not statistically different from the prevalence (8.9%) at 400 m.²⁰

Forrer et al.²¹ described a higher prevalence of Strongyloides infection in older age groups in a study involving more than 2,500 subjects from 60 communities in Cambodia. In this study, subjects older than 49 years were 60% more likely to have strongyloidiasis. Other recent studies in Angola and Lao People’s Democratic Republic (PDR) showed an association between infection and age.^6,22 Similarly, in our study, strongyloidiasis was associated with increasing age. Strongyloides stercoralis has a complex life cycle, including an autoinfection phase where larvae mature within the host and start a new cycle without ever reaching the environment. Autoinfection allows Strongyloides to remain in the host for prolonged periods that may be counted in decades.²³ In elderly patients, age-related malnutrition and impaired immunity can increase susceptibility to severe forms of strongyloidiasis in endemic areas.^24,25 Case reports of strongyloidiasis in the elderly causing hyperinfection syndrome, invasive enteritis, and disseminated infection have resulted in high mortality.^24,26,27

In the community, strongyloidiasis causes only few and unspecific symptoms which rarely prompt medical consultation or diagnostic workup.²⁸ In our study, few subjects with strongyloidiasis reported symptoms, and in most cases, the prevalence of these was not different from the symptoms reported by subjects without the infection. This contrasts with a study by Khieu et al. among Cambodian children where almost a third of those infected reported diarrhea and 10% reported itchy skin within the previous 2 weeks. Children with strongyloidiasis were significantly more likely to report these symptoms than uninfected children.²⁹ As with other soil-transmitted helminth infections, it is likely that subjects with the highest parasite burdens report symptoms more often. However, assessing Strongyloides burden of infection in individuals is difficult as no quantitative tests are readily available.

Strongyloides infection was associated with socioeconomic characteristics in our study population. These included living under a tin roof, owning poultry, use of the municipal sewage system, and having health insurance. More subjects with Strongyloides reported living under a tin roof than subjects without Strongyloides, and fewer subjects with Strongyloides reported owning poultry than subjects without Strongyloides. These characteristics suggest a lower socioeconomic status among subjects in these rural farming communities. Recent studies have described the association between lower socioeconomic status and Strongyloides infection.^21,30 Of note, using the municipal sewage system for excreta elimination was associated with a higher prevalence of Strongyloides infection in our study. This contrasts with a study by Echazu et al.³¹ that showed a 2-fold increase in the risk of strongyloidiasis among subjects without access to improved sanitation. A small study in Argentina showed a decrease in incident Strongyloides infections among subjects who moved to houses with improved water and sanitation compared with those with no access to these facilities.³² Studies in South-East Asia show that the use of improved latrines compared with open defecation was associated with a lower risk of infection.²⁹ However, earlier studies have shown inconsistent results with the use of improved sanitation. Hall et al.³³ reported that the use of community latrines was associated with a higher prevalence of strongyloidiasis than owning a latrine. Yori et al.⁸ studied 800 subjects in a rural community from the Peruvian Amazon and failed to find an association between methods of feces disposal and strongyloidiasis. In our study, feces were observed in the floor around the toilet bowls during the household visits in Quellouno district. Although, it is unclear if this can explain our findings, it suggests that environmental contamination may have existed in the bathroom floor. Adults or children used to other types of excreta elimination may have problems adapting to improved methods like toilet bowls. This highlights the needs for education of the population when interventions to improve sanitation involve a shift in cultural norms and uses. An alternative explanation could be that defects in the integrity or treatment of the municipal sewage system caused contamination of the environment.

Our study showed that those who had government health insurance were less likely to have strongyloidiasis. Similarly, Forrer et al.²¹ found that another marker of health-care access, distance traveled to the health-care facility, was associated with a higher likelihood of Strongyloides and hookworm coinfection in Cambodia. Liu and Lu studied health inequalities at the community level in Rwanda and found that health insurance and travel time to the health-care facility were the two major contributors to health inequalities and medical care utilization, especially among the poorest groups.³⁴ Thus, access to health care may also play a role in the epidemiology of Strongyloides in rural areas.

Walking barefoot and bathing in rivers/streams were associated with Strongyloides infection in our study. Other authors have reported similar results. Herrera et al.³⁵ in a small case–control study in Peru found that participants who bathed in the river 3–4 times/week were more likely to have strongyloidiasis than those who did not bathed in the river. Senephansiri et al.⁶ reported that not wearing shoes when leaving the house was associated with increased frequency of strongyloidiasis in a small study involving 500 subjects in Lao PDR. Forrer et al. administered Strongyloides treatment and education on hygiene to subjects with the infection and followed them up for 2 years. Subjects who failed to wear shoes at home or when defecating despite the intervention were 2.6 times more likely to acquire strongyloidiasis in the following 12 months.³⁶ Avoiding direct contact of bare skin with contaminated soil may be a target for prevention and control of Strongyloides. However, accomplishing this behavioral shift despite repeated educational efforts showed to be a difficult task in prior studies.³⁶ Other studies targeting soil-transmitted helminths with packages including education, improved sanitation, and biannual albendazole had no effects over Strongyloides prevalence, highlighting the significant challenges ahead if control will be attempted.³⁷

Our study had limitations that included a small sample size, which likely precluded finding some other associated factors such as altitude. In our study, there was a higher prevalence of infection at lower altitude communities than the high-altitude community, but this difference was not statistically significant. Some of the information collected on symptoms and past medical history relied on subject’s recollection of events happening in the previous 3 months which may have affected report. Our cross-sectional design and limited scope of testing (i.e., single stool samples and no environmental samples were collected) may have missed some factors explaining some of our findings.

In conclusion, Strongyloides is a neglected parasite with a high prevalence in some areas of the Cusco region of Peru, including the highlands. Socioeconomic and sanitary conditions were associated with the infection in our population, which calls for integrated government efforts to improve living conditions, educate the population, and create ivermectin mass deworming programs.