• View in gallery

    Egg counts per gram (EPG) of feces for patients upon enrollment into the study. Numbers in parentheses indicate group medians. There were no significant differences in egg counts between image pattern (IP) groups (Kruskal-Wallis test for human immuno-deficiency virus [HIV]-negative patients and the Mann-Whitney test for HIV-positive patients). IPA = normal liver texture; IPB = small degree of fibrosis, but no clear-cut pathology; IP > B = fibrotic pathology.

  • View in gallery

    Serum glutamic oxaloacetic transaminase (SGOT) levels in persons according to schistosomiasis-associated morbidity and human immunodeficiency virus 1 (HIV-1) status. The lines within the boxes are the medians, the ends of the boxes are the 25th and 75th percentiles, and the whiskers represent the ranges. Numbers in parentheses indicate median CD4 cell numbers for patients for whom SGOT levels were determined. For definitions of image patterns, see Figure 1.

  • View in gallery

    Median CD3, CD4, and CD8 cell counts in human immunodeficiency virus 1-negative schistosomiasis patients with various grades of liver fibrosis. IP = image pattern; IPA = normal liver texture; IPB = small degree of fibrosis, but no clear-cut pathology; IPC = “ring echoes” that appear as pipe stems in a perpendicular scan; IPD = a “ruff” around the main portal vein and bifurcation; IPE = “ruff” extends into the liver parenchyma with other patches of fibrosis as well).

  • View in gallery

    Significant correlations between image pattern and CD4 cell numbers in human immunodeficiency virus 1-negative schistosomiasis patients. The horizontal bars show the medians and the error bars show the 25th and 75th percentiles. Statistical analysis was performed using Spearman’s rank correlation. For definitions of image patterns, see Figure 3.

  • 1

    Colley DG, Garcia AA, Lambertucci JR, Parra JC, Katz N, Rocha RS, Gazzinelli G, 1986. Immune responses during human schistosomiasis. XII. Differential responsiveness in patients with hepatosplenic disease. Am J Trop Med Hyg 35 :793–802.

    • Search Google Scholar
    • Export Citation
  • 2

    Buchanan RD, Fine DP, Colley DG, 1973. Schistosoma mansoni: infection in mice depleted of thymus-dependent lymphocytes. II. Pathology: Altered pathogenesis. Am J Pathol 71 :208–218.

    • Search Google Scholar
    • Export Citation
  • 3

    Lucas S, Musallam R, Bain J, Hassounah O, Bickle Q, Doenhoff M, 1980. The pathological effects of immunosuppression of Schistosoma mansoni-infected mice, with particular reference to survival and hepatotoxicity after thymectomy and treatment with antithymocyte serum, and treatment with hydrocortisone acetate. Trans R Soc Trop Med Hyg 74 :633–643.

    • Search Google Scholar
    • Export Citation
  • 4

    Karanja DMS, Colley DG, Nahlen BL, Ouma JH, Secor WE, 1997. Studies on schistosomiasis in western Kenya. I. Evidence for immune-facilitated excretion of schistosome eggs from patients with Schistosoma mansoni and human immunodeficiency virus co-infections. Am J Trop Med Hyg 56 :515–521.

    • Search Google Scholar
    • Export Citation
  • 5

    Richter J, Hatz C, Campagne G, Bergquist NR, Jenkins JM, 2000. Ultrasound in Schistosomiasis: A Practical Guide to the Standardized Use of Ultrasonography for the Assessment of Schistosomiasis-Related Morbidity. Geneva: World Health Organization. (http://www.who.int/tdr/publications/publications/pdf/ultrasound.pdf).

  • 6

    Colley DG, Katz N, Rocha RS, Abrantes W, da Silva AL, Gazzinelli G, 1983. Immune responses during human schistosomiasis mansoni. IX. T-lymphocyte subset analysis by mono-clonal antibodies in hepatosplenic disease. Scand J Immunol 17 :297–302.

    • Search Google Scholar
    • Export Citation

 

 

 

 

SHORT REPORT: EVALUATION OF HEPATIC FIBROSIS IN PERSONS CO-INFECTED WITH SCHISTOSOMA MANSONI AND HUMAN IMMUNODEFICIENCY VIRUS 1

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  • 1 Center for Vector Biology and Control Research, Kenya Medical Research Institute, Kisumu, Kenya; Center for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya; Ministry of Health, Nairobi, Kenya; Department of Zoology, Kenyatta University, Nairobi, Kenya; Center for Tropical and Emerging Global Diseases and the Department of Microbiology, University of Georgia, Athens, Georgia; Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

To investigate whether infection with human immunodeficiency virus 1 (HIV-1) affects fibrosis development in patients infected with Schistosoma mansoni, we evaluated schistosomiasis-induced pathology in the livers of Kenyan patients co-infected with HIV-1. Compared with persons with schistosomiasis alone (n = 58), there were no significant differences in distribution of ultrasound-detectable pathology in persons with HIV-1 co-infection (n = 23). Similarly, serum aspartate aminotransferase levels were not significantly different in HIV-1+ individuals. Hepatic fibrosis was associated with significantly decreased CD4+ T cell counts, even in the absence of HIV-1 infection. These data suggest that HIV-1 co-infection does not significantly alter the proportion of patients experiencing schistosomiasis-induced fibrosis, but pathology associated with S. mansoni infections leads to CD4+ T cell reductions and thereby may exacerbate the effects of HIV-1 in co-infected individuals.

Granuloma formation and pathology development during schistosomiasis is dependent on, and mediated by, CD4+ T cells responding to egg antigens. In some patients, excessive inflammation leads to deposition of connective tissue and hepatic or bladder fibrosis, depending on the infecting species of schistosome. Schistosomiasis patients with different clinical forms of the disease show different cellular immune response profiles in vitro and regulation of cellular immune responses has been hypothesized to affect the outcome of clinical disease.1 Although development of severe pathology is associated with poorly regulated CD4 cell hyper-responsiveness, the absence of these cells is also associated with morbidity and mortality in experimental models.2,3 T cell-deficient animals infected with schistosomes make smaller granulomas, but experience increased hepatocellular damage as measured by elevated plasma transaminase levels.3 Thus, either an excess or absence of CD4 cell reactivity may increase host morbidity during schistosomiasis. In addition, we have found that human immunodeficiency virus 1 (HIV-1)-infected schistosomiasis patients secrete fewer eggs than their HIV-1-negative counterparts, despite similar water contact exposures and comparable levels of circulating adult worm antigen.4 Lowered numbers of eggs in feces despite infection with similar numbers of adult worms may mean that the eggs that are not excreted become lodged elsewhere in the body, possibly in the liver. With schistosomiasis and HIV now commonly occurring together in sub-Saharan Africa, it is important to investigate the implications of HIV co-infection and the resultant diminution of CD4+ T cells on development of hepatic fibrosis and/or toxicity.

We have been investigating a cohort of car washers with intense Schistosoma mansoni infections in Kisumu, Kenya, along the shores of Lake Victoria, for the last several years. This study was designed to determine whether HIV-1 co-infection has any bearing on schistosomiasis-related pathology. Specifically, we wished to determine whether HIV-1 co-infection alters the proportion of persons with schistosomiasis-induced liver fibrosis or plasma levels of enzymes associated with hepatocellular damage. We also evaluated CD3, CD4, and CD8 cell counts in persons with and without schistosomiasis liver pathology.

The study protocol was reviewed and approved by the institutional review boards of the Centers for Disease Control and Prevention, according to the guidelines of the U.S. Department of Health and Human Services, and the Kenya Medical Research Institute. Study participants included occupationally exposed car washers greater than 18 years of age with documented water contact. Following informed consent, study participants were offered confidential pretest and post-test HIV counseling by qualified personnel and tested for antibodies to HIV-1 using the Uni-Gold HIV (Trinity Bio-tech, Bray, Ireland) and Determine (Abbott Laboratories, Abbot Japan, Ltd., Tokyo, Japan) test kits according to manufacturer’s specifications. The modified Kato Katz technique (Helm TechR Pesquisa e Desenvolvemento Ltd., Belo Horizonte, Brazil) was used to quantify S. mansoni eggs and other helminth ova. As an assessment of schistosomiasis morbidity, a portable Aloka SSD-620 ultrasound machine with a 3.5 megahertz convex probe (Aloka Co., Ltd., Tokyo, Japan) was used to evaluate schistosome-induced pathology in the liver according to the Niamey classification.5 Based on the degree of liver fibrosis, patients were assigned an image pattern (IP). Patients with a normal liver texture are classified as IPA; IPB is indicative of patients with a small degree of fibrosis, but no clear-cut pathology. We considered persons with an IP > B as having fibrotic pathology. Persons with IPC show “ring echoes” that appear as pipe stems in a perpendicular scan. A “ruff” around the main portal vein and bifurcation characterizes IPD. In IPE, this “ruff” extends into the liver parenchyma with other patches of fibrosis as well. None of our patients showed IPF, in which the bands of fibrosis extend from the main stem and bifurcation extends to the surface of the liver.5 Ultrasound was also used to measure portal vein, left portal branch wall, and gall bladder wall thickness. Ultrasound plus a physical examination were used to detect enlargement of liver or spleen and presence of ascites.

Approximately 30% of the schistosomiasis patients on whom ultrasonography was performed were also positive for HIV-1. Fecal egg counts for patients grouped according to HIV-1 serostatus and IP are shown in Figure 1. In the HIV-1-negative group, 23.1% (12 of 52) or the patients displayed an IP > B, compared with 17.4% (4/23) in the HIV-1-positive group (Table 1). This difference was not statistically significant (odds ratio [OR] = 0.70, P = 0.76, by Fisher’s exact test). Even if the IPB patients are included in the group considered to have abnormal livers, the comparison with a sample population of this size did not achieve statistical significance (OR = 0.46, P = 0.28). Thus, among these patients, persons with HIV-1 were not either more or less likely to experience schistosomiasis-associated liver fibrosis than were HIV-1-negative persons.

Because liver fibrosis in schistosomiasis requires several years to develop, and considering age-prevalence curves associated with both infections, co-infected individuals were probably first infected with schistosomes and may have already developed fibrosis prior to their becoming HIV-1 positive. As a result, the failure to observe a significant difference between HIV-1 infection groups is not surprising. Our data are unable to predict what may occur if a person were infected with HIV-1 and had experienced a marked decrease in their CD4 + T cell levels prior to becoming infected with S. mansoni.

To determine if the presence of schistosome-induced liver pathology and/or HIV-1 infection has any effect on the release of glutamic oxaloacetic transaminase (GOT), an aspartate aminotransferase (AST) which is indicative of liver parenchyma damage, the plasma levels of GOT were quantified in sera from patients by measuring GOT activity using a commercial kit according to manufacture’s protocol (Sigma Chemical Co., St. Louis, MO). Because HIV infection reduces a patients’ CD4 cell count, we hypothesized that it may also alter their ability to form granulomas, perhaps resulting in hepatotoxicity and increased GOT/AST as demonstrated in experimental schistosome infections of T cell-deficient mice.2,3 The levels of GOT in plasma of schistosomiasis patients with hepatic fibrosis (n = 11) were within normal levels (< 40 U/L), were not different from those of persons without hepatic fibrosis (n = 19), and were not affected by HIV co-infection (21 patients over a wide range of CD4 + T cell counts). No significant differences were observed across IPs or between patients with equivalent IPs but differing in HIV-1 status (Figure 2). Similarly, there were no significant correlations between CD4 + T cell counts and GOT levels in either HIV-1-positive or HIV-1-negative individuals. Usually, schistosomiasis patients (even those with hepatosplenic disease) do not have elevated levels of GOT/AST because the granulomatous response does not damage the parenchyma. However, immunocompromised mice with a decreased ability to form granulomas have elevated GOT/AST levels in their sera as a result of proteases released from the eggs. The difference in observations between humans and mice may reflect disparities in CD4 + T cell reduction or the relative parasite loads of the two hosts. Immunocompromised mice have both a complete depletion of CD4 + T cells as well as a much higher ratio of parasite burden to host size than even the most highly infected humans. Nevertheless, at least at the levels of CD4 + T cells in these patients, co-infection with HIV-1 did not appear to render their livers more susceptible to damage by egg proteases.

With the prediction that any reduction in fibrosis or increase in plasma GOT levels among HIV-1-positive individuals would be accompanied by reduced CD4 + T cell counts, we also determined mean absolute CD3, CD4, and CD8 counts using the FACScount instrument and reagents (Becton Dickinson, San Jose, CA) according to manufacturer’s instructions (Figure 3). Interestingly, in addition to observing reduced CD4 + T cell counts in peripheral blood of HIV-1-positive individuals, we also found that HIV-1-negative persons with ultrasound-detectable fibrosis demonstrated lower CD4 + T cell counts. These findings are consistent with previous observations that persons with severe hepatosplenic disease display markedly reduced peripheral blood T cells,6 but we had not anticipated that the reduction in CD4 cells would correlate with each increasing grade of liver fibrosis (P = 0.016, by Spearman’s rank correlation, Figure 4). We do not know if the reduced levels in circulating CD4 + T cells are the result of recruitment of circulating cells into the cellular granulomas in the liver or some mechanism by which these cells are destroyed. However, these results suggest that schistosomiasis-associated fibrosis could potentially further exacerbate the depletion of CD4 + T cells in persons co-infected with HIV-1.

Table 1

Comparison of schistosomiasis morbidity by human immunodeficiency virus 1 (HIV-1) status*

HIV-1 negative (n = 58)HIV-1 positive (n = 23)
* IP = image pattern; IPA = normal liver texture; IPB = small degree of fibrosis, but no clear-cut pathology; IPC = “ring echoes” that appear as pipe stems in a perpendicular scan; IPD = a “ruff” around the main portal vein and bifurcation; IPE = “ruff” extends into the liver parenchyma with other patches of fibrosis as well.
IPA4019
IPB60
IPC74
IPD20
IPE30
Enlarged
    Liver12
    Spleen32
Liver and spleen21
Dilated
    Left portal branch wall93
    Portal vein71
Gall bladder wall thickening65
Ascites10
Figure 1.
Figure 1.

Egg counts per gram (EPG) of feces for patients upon enrollment into the study. Numbers in parentheses indicate group medians. There were no significant differences in egg counts between image pattern (IP) groups (Kruskal-Wallis test for human immuno-deficiency virus [HIV]-negative patients and the Mann-Whitney test for HIV-positive patients). IPA = normal liver texture; IPB = small degree of fibrosis, but no clear-cut pathology; IP > B = fibrotic pathology.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 6; 10.4269/ajtmh.2004.71.783

Figure 2.
Figure 2.

Serum glutamic oxaloacetic transaminase (SGOT) levels in persons according to schistosomiasis-associated morbidity and human immunodeficiency virus 1 (HIV-1) status. The lines within the boxes are the medians, the ends of the boxes are the 25th and 75th percentiles, and the whiskers represent the ranges. Numbers in parentheses indicate median CD4 cell numbers for patients for whom SGOT levels were determined. For definitions of image patterns, see Figure 1.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 6; 10.4269/ajtmh.2004.71.783

Figure 3.
Figure 3.

Median CD3, CD4, and CD8 cell counts in human immunodeficiency virus 1-negative schistosomiasis patients with various grades of liver fibrosis. IP = image pattern; IPA = normal liver texture; IPB = small degree of fibrosis, but no clear-cut pathology; IPC = “ring echoes” that appear as pipe stems in a perpendicular scan; IPD = a “ruff” around the main portal vein and bifurcation; IPE = “ruff” extends into the liver parenchyma with other patches of fibrosis as well).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 6; 10.4269/ajtmh.2004.71.783

Figure 4.
Figure 4.

Significant correlations between image pattern and CD4 cell numbers in human immunodeficiency virus 1-negative schistosomiasis patients. The horizontal bars show the medians and the error bars show the 25th and 75th percentiles. Statistical analysis was performed using Spearman’s rank correlation. For definitions of image patterns, see Figure 3.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 6; 10.4269/ajtmh.2004.71.783

Authors’ addresses: Pauline N. M. Mwinzi and Diana M. S. Karanja, Vector Biology and Control Research Centre, Kenya Medical Research Institute, PO Box 1578, Kisumu, Kenya, E-mails: pmwinzi@kisian.mimcom.net and dkaranja@kisian.mimcom.net. Ir-eri Kareko, Centre for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya. Phillip W. Magak, Ministry of Health, Nairobi, Kenya. Alloys S. S. Orago, Department of Zoology, Kenyatta University, Nairobi, Kenya. Daniel G. Colley, Center for Tropical and Emerging Global Diseases, Room 623, Biological Sciences Building, University of Georgia, Athens, GA 30602, E-mail: dcolley@uga.edu. W. Evan Secor, Division of Parasitic Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway, Mail-stop F-13, Atlanta, GA 30341, E-mail: was4@cdc.gov.

Acknowledgments: This paper is published with the permission of the Director of the Kenya Medical Research Institute. We thank Julius Andove and Kenedy Matunda for field and laboratory technical assistance.

Financial support: This project was funded in part by National Institutes of Health grant AI-053695 and The University of Georgia Research Foundation. Pauline N. M. Mwinzi was supported by World Health Organization/Tropical Disease Research training grant no. 971183.

REFERENCES

  • 1

    Colley DG, Garcia AA, Lambertucci JR, Parra JC, Katz N, Rocha RS, Gazzinelli G, 1986. Immune responses during human schistosomiasis. XII. Differential responsiveness in patients with hepatosplenic disease. Am J Trop Med Hyg 35 :793–802.

    • Search Google Scholar
    • Export Citation
  • 2

    Buchanan RD, Fine DP, Colley DG, 1973. Schistosoma mansoni: infection in mice depleted of thymus-dependent lymphocytes. II. Pathology: Altered pathogenesis. Am J Pathol 71 :208–218.

    • Search Google Scholar
    • Export Citation
  • 3

    Lucas S, Musallam R, Bain J, Hassounah O, Bickle Q, Doenhoff M, 1980. The pathological effects of immunosuppression of Schistosoma mansoni-infected mice, with particular reference to survival and hepatotoxicity after thymectomy and treatment with antithymocyte serum, and treatment with hydrocortisone acetate. Trans R Soc Trop Med Hyg 74 :633–643.

    • Search Google Scholar
    • Export Citation
  • 4

    Karanja DMS, Colley DG, Nahlen BL, Ouma JH, Secor WE, 1997. Studies on schistosomiasis in western Kenya. I. Evidence for immune-facilitated excretion of schistosome eggs from patients with Schistosoma mansoni and human immunodeficiency virus co-infections. Am J Trop Med Hyg 56 :515–521.

    • Search Google Scholar
    • Export Citation
  • 5

    Richter J, Hatz C, Campagne G, Bergquist NR, Jenkins JM, 2000. Ultrasound in Schistosomiasis: A Practical Guide to the Standardized Use of Ultrasonography for the Assessment of Schistosomiasis-Related Morbidity. Geneva: World Health Organization. (http://www.who.int/tdr/publications/publications/pdf/ultrasound.pdf).

  • 6

    Colley DG, Katz N, Rocha RS, Abrantes W, da Silva AL, Gazzinelli G, 1983. Immune responses during human schistosomiasis mansoni. IX. T-lymphocyte subset analysis by mono-clonal antibodies in hepatosplenic disease. Scand J Immunol 17 :297–302.

    • Search Google Scholar
    • Export Citation
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