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Objectives To determine the prevalence of intestinal helminth infections in active tuberculosis patients and their healthy household contacts and to assess its association with active TB in an area endemic for both types of infections.
Methods Smear-positive pulmonary TB patients and healthy household contacts were tested for intestinal helminths using direct microscopy and the formol-ether concentration techniques. Three consecutive stool samples were examined before the start of TB chemotherapy. Sputum microscopy was done using the sodium hypochlorite concentration techniques. Participants were also tested for HIV by commercial sandwich enzyme linked immunosorbent assay.
Results The study population consisted of 230 smear-positive TB patients and 510 healthy household contacts. The prevalence of intestinal helminths was 71% in patients and 36% in controls. HIV seroprevalence was significantly higher in patients than in controls (46.7%vs. 11.6%, P < 0.001). Conditional logistic regression analysis showed a strong association between TB and intestinal helminth infection (OR = 4.2, 95% CI 2.7–5.9, P < 0.001), and between TB and HIV infection (OR = 7.8, 95% CI 4.8–12.6, P < 0.0001). The odds of being a TB patient increased with the number of helminth species per person: in individuals with mono-infection it was 4.3 (95% CI 2.8–6.8); in people infected with two species was 4.7 (95% CI 2.5–8.7), and in patients infected with three or more helminths was 12.2 (3.9–52.6).
Conclusion Intestinal helminth infection may be one of the risk factors for the development of active pulmonary TB in addition to HIV infection. This finding may have important implications in the control of TB in helminth endemic areas of the world.
Objectifs Déterminer la prévalence de l'infection intestinale par des helminthes chez les patients ayant une tuberculose (TB) active et chez leurs contacts familiaux sains pour évaluer son association avec une TB active dans une région endémique pour les deux infections.
Méthodes Des patients TB à microscopie positive et leurs contacts familiaux sains ont été testés pour la présence d'helminthes intestinaux au moyen de la microscopie directe et de la technique de concentration au formol-ester. Trois échantillons consécutifs de selles ont été collectés avant le début du traitement contre la TB. La microscopie des crachats a été réalisée en utilisant la technique de concentration basée sur l'hypochlorite de sodium. Les participants ont été testés pour le VIH avec une technique ELISA commerciale standard.
Résultats La population étudiée comprenait 230 patients TB à frottis négatifs et 510 contacts familiaux sains. La prévalence d'helminthes intestinaux était de 71% chez les patients et 36% chez les contrôles. La séroprévalence VIH était significativement plus élevée chez les patients que chez les contrôles (46,7% vs 11,6%, P < 0.001). L'analyse de régression logistique conditionnelle a révéler une forte association entre la TB et l'infection intestinale aux helminthes (OR = 4,2; IC95%: 2,7–5,9; P < 0.001) et entre la TB et l'infection VIH (OR = 7,8; IC95%: 4,8–12,6; P < 0.0001). Les Odd Ratios (OR) pour la TB augmentent avec le nombre d'espèces d'helminthes par personne; chez les individus avec une monoinfection (OR = 4,3; IC95%: 2,8–6,8), chez les individus infectes par deux espèces d'helminthes (OR = 4,7 IC95%: 2,5–8,7) et chez les individus avec trios espèces d'helminthes ou plus (OR = 12,2; IC95%: 3,9–52,6).
Conclusion En plus de l'infection VIH, l'infection intestinale aux helminthes pourrait aussi être un des facteurs de risque pour le développement de la TB pulmonaire active. Cette observation pourrait avoir des implications importantes dans le contrôle de la TB dans les régions du monde endémiques pour les helminthes.
Objetivos Determinar la prevalencia de infecciones intestinales por helmintos en pacientes con tuberculosis activa y sus contactos familiares sanos, así como su asociación con la TB activa, en un área endémica para ambos tipos de infecciones.
Métodos Se determinó la presencia de helmintos intestinales en pacientes con tuberculosis pulmonar, con lámina positiva, y sus contactos familiares sanos mediante microscopía directa y la técnica de concentración de formol-éter. Se examinaron tres muestras fecales consecutivas antes de iniciar la quimioterapia para la TB. Se realizó microscopía de esputo utilizando la técnica de concentración del hipoclorito de sodio. Los participantes también fueron examinados para VIH mediante un ensayo comercial inmunoenzimático de tipo sandwich.
Resultados La población de estudio consistió en 230 pacientes tuberculosos con lámina positiva y 510 contactos sanos. La prevalencia de helmintos intestinales fue del 71% en los pacientes y del 36% en los controles. La seroprevalencia de VIH fue significativamente mayor en los pacientes que en los controles (46.7% vs 11.6%, P < 0.001). Un análisis de regresión logística condicional mostró una asociación fuerte entre la TB y la infección por helmintos (OR = 4.2, 95% IC 2.7–5.9, P < 0.001), y entre infección por TB y VIH (OR = 7.8, 95% IC 4.8–12.6, P < 0.0001). El riesgo relativo indirecto (odds) de ser un paciente de TB aumentaba con el número de especies de helmintos por persona: en individuos con mono infección era 4.3 (95% IC 2.8–6.8); en personas infectadas con dos especies, era 4.7 (95% IC 2.5-8.7), y en pacientes infectados con 3 o más helmintos el riesgo relativo era de 12.2 (3.9–52.6).
Conclusión La infección por helmintos intestinales puede ser uno de los factores de riesgo asociados al desarrollo de TB pulmonar activa, además de la infección por VIH. Este hallazgo puede tener implicaciones importantes en el control de la TB en áreas endémicas para helmintos.
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- Patients and methods
Mycobacterium tuberculosis, the agent that causes tuberculosis, is estimated to infect about one-third of the world population (WHO 2002). Only a small fraction of the infected individuals develop the disease and the vast majority remains disease free. The outcome of infection depends on the cell-mediated immunity. Macrophages infected with mycobacteria interact with both CD4+ and CD8+ T cells inducing the release of cytokines, which leads to macrophage activation and, in most cases, control of the infection (Chan & Kaufmann 1994). The reason for variation in individual susceptibility to tuberculosis is not fully understood. It is generally accepted that protection against tuberculosis is strongly associated with enhanced Th1 cell mediated immune responses (Cooper & Flynn 1995) while susceptibility to the disease is associated with reduced Th1 type responses and/or enhanced Th2 responses with high interleukin 4 (IL-4), IL-5 and IL-10 (Schaible et al. 1999). Th2 responses are usually elicited by helminth infections (Abbas et al. 1996). Th1 and Th2 cells were shown to negatively influence each other both in vitro and in vivo: IFN-γ inhibits Th2 responses while IL-4 inhibits Th1 responses.
It is becoming increasingly clear that helminth infections, in addition to stimulating vigorous Th2 responses, can induce suppressive T cell populations known as regulatory T cells (Tregs). Tregs produce inhibitory cytokines (IL-10 and TGF-β) that suppress Th1 type responses and interfere with effector T cell activation (Shevach et al. 2001). Initially it was thought that Tregs are important primarily in the control of pathogenic T cells and autoimmune responses. Recently however, it has become apparent that Tregs could be induced to regulate responses to pathogens (Iwashiro et al. 2001; Kullberg et al. 2002; Satoguina et al. 2002; Hesse et al. 2004; Maizels et al. 2004). Reports have shown the involvement of Tregs in helminth induced immunosuppression (King et al. 1993; Doetze et al. 2000; Satoguina et al. 2002; Pearce & McKee 2004). In experimental nematode infection, it has been observed that there was enhancement of Treg function associated with up regulated Th2 responses (Gillan & Devaney 2005). Such enhanced Treg function associated with helminth infection may suppress Th1 responses directed against unrelated antigens and/or pathogens (Oldenhove et al. 2003).
However, the impact of worm-induced Th2 and/or Treg responses on protection against mycobacterial diseases or on vaccinations against intracellular pathogens in general remains to be investigated (reviewed by Rook et al. 2005). These are questions of important public health concern particularly in the developing world where both infections are common.
In an effort to address this, we analysed the cellular responses to TB antigens of T cells from worm-infested but clinically healthy adolescent Ethiopians. We found that worm infection impairs TB antigen specific cellular responses (Elias et al. 2001). Moreover, in animal models we observed that the protective efficacy of BCG vaccination against TB was significantly lower in Schistosoma mansoni pre-infected mice (Elias et al. 2005a). These findings indicate the importance of chronic worm infections on mycobacterial immunity. In the present work we conducted a case control study to examine the association between intestinal helminths infection and active tuberculosis.
Patients and methods
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- Patients and methods
This study was conducted between October 1999 and January 2002 in a teaching hospital of Gondar University, North West Ethiopia, where the incidence of TB is estimated at 292/1 00 000 (WHO 2002) and where infection with intestinal worms is frequent (Jemaneh 1998). All patients had clinical features consistent with TB and positive sputum smear microscopy. The study was explained to all smear positive TB patients diagnosed at the medical outpatient department of the Gondar University Hospital in the study period. Patients who volunteered to take part during the study were registered as study subjects. Controls were selected from patients’ households based on the following criteria: residence in the same house as the patient for the past 12 months; apparently healthy upon physical examination and free of signs and symptoms of TB; giving written informed consent to take part in the study.
Sputum smear microscopy was done using the sodium hypochlorite concentration technique (Gebre et al. 1995). Smear positivity was defined as two of three morning sputum samples positive for acid-fast bacilli. Smear positive TB patients were treated based on the standardized short course chemotherapy with isoniazid, rifampicin, pyrazinamide and ethambutol (or streptomycin) during the first 2 months (intensive phase) followed by isoniazid and ethambutol for 6 months.
HIV serology was done by enzyme linked immunosorbant assay (Enzygnost, Boering, Germany) and a confirmatory test was performed on all samples by another person unaware of the results of the first test using another commercially available HIV ELISA kit obtained from a different company (Vironstica, Baxtel, The Netherlands). Three samples were collected on three consecutive days and parasitologically examined on the same day by direct microscopy and formol-ether concentration (Cheesbrough 1998). Every 10th specimen was reexamined for quality control and a discrepancy of 10% or less for the whole study was regarded acceptable. The initial examination was performed by a senior laboratory technician experienced in microscopic identification of intestinal parasites while the quality control examination was performed by an experienced parasitologist without the knowledge of the results from the initial examination. A subject was labelled helminth positive when any of the three samples were found to have egg or larvae of parasites. All three stool samples were collected before the initiation of anti-TB therapy.
The association between TB and the variables helminth infection status, HIV status, sex and age was evaluated using conditional logistic regression analysis. The variables were included in the conditional logistic regression because they were considered potential confounders. The association was expressed as odds ratio (OR). The difference in helminth infection rate between the two groups was examined using the x2 test.
The study had received ethical approval from the Gondar University, College of Health Sciences Research Committee. Informed written consent was obtained from all volunteers (or legal guardians of participants under the age of 18) enrolled in this study. Appropriately trained physicians conducted pre and post HIV test counselling to all volunteers who took part in this study. Antiretroviral treatment was not available in the country at the time of this study and therefore was not offered to HIV positive volunteers. All subjects who were found to have helminth infection were given appropriate treatment.
The data was double entered and cross-checked using Epi-Info Version 6 software (Centers for Disease Control and prevention, Atlanta, GA, USA). Statistical analysis was performed using STATA Version 7 (STATA Corporation, College Station, TX, USA) statistical software. The prevalence of intestinal helminths and HIV infection was compared between the groups using the x2 test. Conditional logistic regression analysis was used to assess the association between being TB patient and intestinal parasite infection, HIV infection, age, and sex. A P value <0.05 was considered statistically significant in all analyses.
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A total of 312 smear positive TB patients and 754 household contacts of the cases were invited to take part in the study. Of 312 smear positive TB patients diagnosed at Gondar Hospital during the study period, 82 were unwilling to take part in the study. Of 754 controls, 37 were excluded because they had symptoms suggestive of TB but were smear negative; a further 207 were excluded because they did not consent to HIV testing. The study included 230 smear positive TB patients and 510 controls. The mean age of the TB cases was similar to that of the controls (Table 1). Age was missing for seven of the TB cases and 10 of the control subjects. A 52.6% of the cases and 55.5% of the controls were males (Table 1).
Table 1. Gender and age of TB patients and controls
|Groups||Sex|| n ||Age|
Helminth species prevalent in the study population include: Ascaris lumbricoides, Hookworm, Strongloides stercoralis, Trichuris trichiura, S. mansoni and Enterobius vermicularis (Table 2). Neither in the TB patients nor in the healthy household contacts was the presence of intestinal worms linked to any worm-specific clinical symptoms.
Table 2. Prevalence of intestinal helminths and HIV1/2 in TB patients and their healthy household contacts
|Type of infection||Group|
|TB patients||Controls||OR (95% CI)|
| n (%)|| n (%)||Crude||Adjusted|
| A. lumbricoides ||123 (53.5)||101 (19.8)||5.6 (3.7–8.5)||5.7 (3.6–8.9)|
|Hookworm||65 (28.3)||65 (12.7)||3.0 (1.9–4.9)||3.3 (2.0–5.5)|
| S. stercoralis ||36 (15.6)||20 (3.9)||2.1 (1.1–4.1)||2.4 (1.2–5.1)|
| T. trichuria ||10 (4.3)||26 (5.1)||0.7 (0.3–1.7)||0.6 (0.2–1.5)|
| S. mansoni ||9 (3.9)||31 (6.1)||0.3 (0.02–1.3)||0.5 (0.02–1.0)|
| E. vermicularis ||0 (0.0)||6 (1.2)||ND||ND|
|Number of worm species per subject|
|1||103 (44.8)||131 (25.7)||3.5 (2.4–5.1)||4.3 (2.8–6.8)|
|2||47 (20.4)||48 (9.4)||4.9 (2.9–8.4)||4.7 (2.5–8.7)|
|3+||13 (5.7)||6 (1.2)||11.2 (3.9–32.0)||12.2 (3.9–52.6)|
|Any worm infection||163 (70.9)||185 (36.3)||4.1 (2.8–5.8)||4.2 (2.7–5.9)|
|HIV1/2||108 (46.9)||60 (11.8)||7.7 (4.8–12.5)||7.8 (4.8–12.6)|
Of the 230 smear positive TB patients included in the study, 70.9% (163/230) tested positive for one or more intestinal helminths; 36.3% (185/510) of the controls did. The difference was statistically significant (P < 0.001). The proportions of TB patients infected with 1, 2, 3 or more species of worms were 44.8% 20.4% and 5.7%, respectively; in controls these proportions were 25.7%, 9.4% and 1.2% (Table 2). A 108 (46.9%) TB patients and 60 (11.8%) controls were HIV infected. The difference was statistically significant (P < 0.001). Double infection with both intestinal helminths and HIV was found in 33% (76/230) of TB patients and 4.5% (23/510) of the controls. While the prevalence of worms in the different age groups was comparable in the controls, in patients there were age-related differences in worm infection rate: it was highest in the age group 10–19 and lowest in the over 50 age group (Table 3).
Table 3. Distribution of helminth infection in the different age groups and in both sexes of TB patients and controls
|Infected||OR* (95% CI)||Infected||OR* (95% CI)|
| 10–19||19 (86.4)||1||34 (37.4)||1|
| 20–29||82 (82.0)||0.7 (0.2–2.5)||46 (35.4)||0.9 (0.5–1.6)|
| 30–39||30 (55.6)||0.2 (0.05–0.7)||48 (45.7)||1.3 (0.7–2.4)|
| 40–49||16 (80.0)||0.6 (0.1–3)||28 (34.6)||0.8 (0.5–1.6)|
| ≥50||16 (47.1)||0.1 (0.02–0.5)||32 (31.1)||0.7 (0.4–1.3)|
| Female||87 (79.8)||1||77 (33.9)||1|
| Male||76 (62.8)||0.9 (0.5–1.6)||108 (39.2)||1.2 (0.9–1.8)|
The prevalence of worm infection in HIV-negative TB patients (71.3%; 87/122) was similar to that of HIV-positive patients (70.4%; 76/108; P = 0.5). Worm infection rates in HIV-positives (40%; 24/60) and HIV-negative controls (35.8%; 161/450) were comparable (P = 0.8). Regression analysis to assess the association between individual helminth species and HIV status found no significant association between HIV status and any of the helminth species observed in this study (result not shown).
Conditional logistic regression analysis revealed a strong association between TB and HIV infection (adjusted OR 7.8, 95% CI 4.8–12.6, P < 0.001) and between TB and intestinal helminth infection (adjusted OR 4.2, 95% CI 2.7–5.9, P < 0.0001). The association between helminth infection and active TB increased progressively with the number of helminth species the person carried (Table 2); the odds of being a TB patient for a person with a single species of helminths was 4.3, 95% CI (2.8–6.8); for a person infected with two helminth species it was 4.7, 95% CI (2.5–8.7) and for individuals infected with three or more helminths the odds of being a TB patient increased to 12.2, 95% CI (3.9–52.6). Of the five species of helminths for which regression analysis was performed, A. lumbricoides, hookworm and S. stercoralis were independently and significantly associated with active TB whereas the association between T. trichiura or S. mansoni infection and active TB was not significant (Table 2).
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Several investigators have shown that an established immune profile against a given infection could affect the outcome of subsequent infections or vaccinations (Curry et al. 1995; Helmby et al. 1998). Our studies in the mouse model have shown that S. mansoni infection impairs resistance against subsequent infection by Mycrobacterium bovis BCG (Elias et al. 2005b). However, whether a similar situation holds true in humans is a question that remains to be addressed. In a preliminary study to address this problem in humans, we have demonstrated that in vitro cellular responses to TB antigens were reduced in individuals with chronic worm infection (Elias et al. 2001). A study in South Africa found the highest incidence of TB in the poorest villages (Beyers et al. 1996). The reason for the concentration of TB in this village cannot solely be attributed to overcrowding and rapid transmission, as there was a preponderance of unique strains of M. tuberculosis suggesting reactivation of latent infection (Warren et al. 1996). The poor communities in this village were highly infested with helminths that induce potent Th2 responses to both parasite and non-parasite antigens. This led Bentwich et al. (1999) to suggest that helminth infection could increase susceptibility to TB. This hypothesis is supported by our finding that TB specific cellular responses were reduced by incident helminth infections in humans (Elias et al. 2001) and that S. mansoni infection in the mouse model reduced the protective efficacy of BCG vaccination against M. tuberculosis (Elias et al. 2005a). Others studies showed an association between intestinal helminth infections in humans and immune activation, defects in signal transduction as well as anergy (Bentwich et al. 1996; Borkow et al. 2000). We therefore hypothesized that chronic worm infection common in developing countries in sub-Saharan Africa could be one of the factors (besides high HIV infection rate, poverty and deprivation) associated with the high incidence of TB.
Tristao-Sa et al. (2002) reported a higher rate of intestinal nematodes in patients with pulmonary TB and Diniz et al. (2001) observed a strong association between intestinal nematode infection and multibacillary leprosy. Using a larger population size in a more controlled study, we showed that intestinal worm infections were more prevalent in active TB patients than in their healthy household contacts. This finding further confirms the importance of chronic worm infections on the pathogenesis of TB.
Considering the fact that TB is a chronic disease, it is difficult to determine whether or not helminth infection preceded the development of active TB. It is possible that having active TB may predispose to helminth infections. However, we think that the immune modulation induced by helminth infection may affect immunity to TB. The presence of intestinal worms may shift the immune background towards a Th2 profile as well as increased IL-10 producing Tregs (van den Biggelaar et al. 2000) which may favour the establishment of mycobacterial infection and the development of active disease. Consistent with this explanation, other reports have shown that development of active TB is associated with enhanced Th2 type immune responses (Lienhardt et al. 2002; Fincham et al. 2003; Talreja et al. 2003) and such responses are protective against helminth parasites (Turner et al. 2003; Bradley & Jackson 2004; Jackson et al. 2004). This would argue against the explanation that having active TB favoured infection with intestinal helminths.
A study determining worm load is needed to evaluate whether individuals with high worm burden have an increased risk of developing TB to further support the hypothesis that worms are one of the risk factors besides HIV infection, for developing active TB in worm-endemic countries. However, we evaluated the association between the number of helminth species a person carried and active TB and showed that the odds of being an active TB patient increased progressively with the number of species of helminth parasites the person carried. This strongly supports the hypothesis that helminth infection may be an important risk factor for the development of active TB.
The association between TB and worm infection observed in this study could be confounded by poverty. Indeed, poverty and malnutrition are important predisposing factors to TB (Diaz de Quijano et al. 2001; Singh et al. 2002; Leung et al. 2004) and it is possible that the poorer people were more likely to get infected with worms and also more likely to get TB. However, the fact that our controls were selected from the same households, living under the same socio-economic conditions as the patients, reduces the possibility that the observed association is confounded by socioeconomic status.
In this study, age and sex were not associated with having active TB. This may appear surprising given the fact that more men than women are diagnosed to have TB worldwide (Austin et al. 2004) and that susceptibility to TB may be higher at extreme ages in parts of the world where HIV infection, the greatest risk factor for developing TB, is low (Pillay et al. 2001). Our study was conducted in an area where the prevalence of HIV in the adult population is high (Demissie et al. 2000; Schon et al. 2003) and hence the epidemiology of TB in such areas may not follow the same pattern as in low HIV prevalence settings. A report from the same area showed that the mean age of smear positive TB patients was about 29 years and there were comparable numbers of males and females in the smear positive TB patients in the area (Schon et al. 2003) consistent with the current data. One may question the sampling in this study as we only included smear positive TB patients who account for only about 35% of all TB cases diagnosed in the area (D. Elias et al., unpublished data). In addition some of those eligible were excluded because they did not volunteer to take part in the study. We must therefore admit that the age and sex distribution of the patient population included in this study may not reflect the situation in the whole patient population in the area.
Demissie et al. (2000) reported a prevalence of HIV in smear positive TB patients in Ethiopia of about 50%. The seroprevalence of HIV in smear positive TB patients in this study was 46.9% compared to 11.8% in the controls. It may be argued that the higher worm infection rate in TB patients could be due to HIV mediated immunosuppression that could predispose patients to getting different types of infections including worms. This is indeed a valid argument. To examine this possibility, we compared worm infection rates in HIV positive and negative controls and found that they were comparable, indicating that the association between worms and TB was not confounded by the high HIV infection rate in the TB patients and that helminths were associated with active TB independently of HIV infection.
In this study significant association with active TB was found only for A. lumbricoides, hookworm and S. stercoralis but not for T. trichiura or S. mansoni. This may appear intriguing given the observations that these helminths could influence immune responses against unrelated infections (Cooper et al. 2000; Doetze et al. 2000; Elias et al. 2001,2005a,b). However, our study was conducted in an area where the overall prevalence of these helminths is low (less than 6%). As a consequence there were too few people positive for T. trichiura or S. mansoni to assess the association with sufficient power. A further clinical study in an area with high prevalence of Schistosoma or Trichuris is required to assess the impact of these worms on the development of active TB.
There might be factors that affect an individual's susceptibility to HIV, TB or helminths that were not measured, for instance differences in household members’ immunization status, occupations, drinking and smoking habits. Therefore, the results of this study should be confirmed by a larger and more controlled study in an area with high incidence of TB.
In summary, the observations in this study indicate a strong association between worm infection and TB in areas endemic for both types of infections. This finding, if supported by a prospective study, has relevance to the control of TB in worm-endemic parts of the world, where regular mass deworming could improve general health and offer an affordable, effective and novel means to reduce the burden of TB.