Anaemia and gastro-intestinal function in ancylostomiasis.
Hookworm infestation is common in India. This paper describes the study of anaemia and gastro-intestinal function in ancylostomiasis.
Fifty four patients between 10 and 55 years, suffering from hookworm infestation were studied. There were 46 males and 8 females. Fifteen of these were less than 20 years while 9 cases were over 40 years of age. Majority of them, (67%), were farmers or farm labourers from adjoining areas of Maharashtra, near Bombay city.
Each patient had a thorough clinical evaluation and routine laboratory studies. In addition, the following investigations were carried out:
(1) Egg count by the method of Stoll.
(2) Bone-marrow morphology (28 cases).
(3) RBC fragility test (5 eases).
(4) Serum iron estimation by the method of Brendrstrup.
(5) Serum vitamin B12 estimation by the method of Ross.
(6) Blood volume, RBC survival, intestinal blood and iron loss estimation by Cr tagged RBC, , ,  (5 cases).
(7) Radioactive B12 absorption by the method of Schilling.
(8) Gastric analysis by augmented histamine test, and fibrogastroscopy.
(9) Glucose tolerance test, the blood glucose being estimated by the method of Folin and Wu.
(10) Lactose tolerance test.
(11) d-Xylose excretion test by the method of Roe and Rice, as modified by Santini et al
(12) Fecal fat estimation by the method of van de Kamer et al.
(13) Per oral intestinal biopsy by Crosby Capsule.
(14) Barium meal X-ray studies.
(15) Serum protein electrophoretic pattern.
(16) Liver function tests and liver biopsy. After these tests the patients were treated for ancylostomiasis and whenever indicated some or all of the above tests were repeated.
Weight loss, generalized weakness, anorexia, exertional dyspnoea, flatulence, bone pains, paresthesia, feverish feeling, gurgling and vague abdominal pain were common symptoms. Diarrhoea and glossitis were complained of by 40% and 29% of patients respectively; constipation was present in 22% of patients.
On physical examination, weight loss; anaemia, poor nutrition, hapatomegaly, pedal edema and engorged neck veins were common findings. Of 7 patients between the ages of 10 and 17 years, 6 were judged to be stunted for their ages.
Stool egg count
The average egg count by the method of Stoll was 278,212 eggs per day with a range of 11,505 to 800,000, eggs per day. Bight of these 54 patients also had ascariasis, 7 had trichuriasis, 6 had amoebiasis and 3 had giardiasis, in addition to ancylostomiasis.
These are shown in [Table 1]
Haemoglobin values: The mean haemoglobin was significantly low in these patients (p < 0.001). Forty five of 54 patients (81%), had a haemoglobin value of less than 10 gm per 100 ml; while only 2 patients had haemoglobin values within normal range. Iron deficiency anaemia was detected in 75% of cases.
(b) Serum iron
The mean serum iron level was nearly the same as that in normal controls probably from previous iron therapy in 8 cases. The serum iron was decreased to less than the lower limit of normal in 18 untreated patients. Seventeen with low serum iron levels had haemoglobin values of less than 7.0 gm per 100 ml.
(c) Serum vitamin B12
The mean and individual serum B12 levels were generally normal or high probably due to prior treatment with vitamin B12
(d) Fragility test
The RBC fragility test was normal in 5 patients in whom it was performed.
(e) Bone-marrow morphology
Bone marrow was normoblastic in 23 and dimorphic in five patients. Of these 5 patients with dimorphic bone marrow, only occasional megaloblasts were seen in 3 patients while in the remaining two, it was frankly megaloblastic. In majority of patients, the bone marrow showed moderate erythroblastic hyperplasia and absent bone-marrow hemosiderin.
(f) Radio chromium studies
[Table 2] shows the results of radio chromium studies. The average total blood volume was decreased in patients with ancylostomiasis as compared to that in the normal controls. The decrease was more marked in red cell mass, the blood volume being compensated by an increase in the plasma volume.
The half life of Cr tagged red cells in 4 control patients was between 20 and26 days [Table 2] in 5 patients with ancylostomiasis, it varied between 14 and 25 days.
The average estimated intestinal blood loss in these patients was 16 ml per day, with a range of 4 to 31 ml per day [Table 2]. The average calculated iron loss through the intestinal tract was 2.66 mg per day with a range of 0.4 to 4.7 mg per day. The average blood loss per worm was 0.22 ml per day with a range of 0.02 to 1.04 ml per day.
(a) Gastric analysis and gastroscopy
Gastric acid output by augmented histamine test using 76 ug/kg of histamine acid phosphate in 28 patients was significantly low both during basal and maximally stimulated states [Table 1]. The appearance of the gastric mucosa on fibrogastroscopy was normal in nine of the 12 patients examined. An antral carcinoma was seen in one. One patient with partial gastrectomy, gastrojejunostomy and ancylostomiasis showed postoperative gastritis, while only one patient with uncomplicated ancylostomiasis showed mild superficial gastritis.
(b) Labelled vitamin B12 absorption test (Schilling's test)
The mean urinary excretion was significantly lower in ancylostomiasis than that in normal controls [Table 1]. Of the 16 patients with abnormal (low) excretion of Co B12, two were suffering from tropical sprue, one from coeliac disease, another had had partial gastrectomy with gastrojejunostomy and 3 had giardiasis which could account for malabsorption of vitamin B12. Two patients with frankly megaloblastic anaemia had low urinary Co B12.
Schilling's test was repeated in 8 patients after they had been dewormed. In 3, it was normal and in 4 abnormal both before and after treatment. Only one patient showed some improvement from 5.6 to 8.7 per cent of administered dose after deworming.
(c) Glucose Tolerance Test
The mean maximum rise over the fasting level in patients with ancylostomiasis was statistically lower than that in controls [Table 1]. Nine of 41 (22 per cent) patients had flat glucose tolerance curve since they failed to show a rise of 30 mg% or more over the fasting level.
(d) Lactose tolerance test
Lactose tolerance test was considered abnormal if the maximum rise over the fasting level was less than 20 mg per 100 ml, and if the patient developed diarrhoea with pain. Using these criteria, none of the patients developed diarrhoea or pain after the loading dose of lactose while only 2 of the 14 patients (14%) showed a maximum rise of less than 20 mg per 100 ml. The mean maximum rise over the fasting level was similar in patients with ancylostomiasis and normal controls [Table 1].
The mean 5 hour urinary excretion after a dose of 5 gm of d-Xylose was significantly lower in patients with ancylostomiasis than that in the normal controls. Eight of these 39 patients (20 per cent) had abnormally low excretion of d-Xylose.
The mean daily fecal fat excretion was similar in patients with ancylostomiasis and normal controls. Six patients with ancylostomiasis had steatorrhoea. Of these, one had tropical sprue, one had coeliac disease, one had had partial gastrectomy with gastrojejunostomy and the remaining three had giardiasis.
(g) Liver function tests and liver biopsy
Twelve of these 54 patients had clinically palpable, enlarged liver while the spleen was palpable in only two cases. Only two patients had abnormal thymol turbidity and cephalin cholesterol flocculation tests. Liver biopsy was normal in 5 patients tested.
The total serum proteins and the electrophoretic pattern were measured in 38 of these patients [Table 1]. Hookworm anaemia was associated with a somewhat lower mean total protein level and significantly lower mean albumin level and raised globulin levels than those in normal controls. Only 2 patients had normal serum albumin levels above 3.5 gm per cent.
(h) Per oral intestinal biopsies
Adequate biopsy specimens (20 from jejunum and 4 from duodenum) were available for analysis in 24 patients. While reviewing the biopsies, particular attention was paid to the general appearance of the villi and to the length, breadth and width of the villi which were measured by an ocular micrometer. Particular attention was also paid to the type of epithelial cells, the type and location of epithelial cell nuclei, the number of goblet cells and the type of cellular in filtrate in the glands and in the lamina propria. Using these criteria, it was observed that the average length of villi in these patients was shorter than that reported from Western countries but similar to our own normal controls. The breadth and thickness were about the same [Table 1]. Clubbing and flattening of the villi were seen in four; partial villous atrophy in three while subtotal atrophy in none. The epithelial cells and their nuclei were judged to be normal in all but three specimens. The average goblet cell count (counted from five villi in each specimen) was 8.8 with a range of 2 to 17 cells. The cellular infiltrate was also within normal range except in three biopsies. Thus, intestinal morphology was normal in all except in 3 specimens which showed partial villous atrophy with clubbing, flattening and abnormal cellular infiltrate.
Thus, even though the mean figures for glucose, d-Xylose, Co B12, were significantly lower than those in normal controls, only about one-fourth of these subjects with ancylostomiasis showed either flat glucose or lactose tolerance or decreased excretion of d-Xylose or Co B12. Moreover, as discussed above, there was some other cause than ancylostomiasis for the presence of this malabsorption.
The symptomatology in our patients was similar to that described by others., , , , , , , , , , 
The incidence of megaloblastic anaemia in ancylostomiasis has been reported to vary from 0-85%., , , , , ,  Of 5 patients with dimorphic anaemia from this series, only two cases showed frankly megaloblastic bone marrow. One of these was suffering from coeliac disease while the other had tropical sprue. Daftary and Bhende, reporting an incidence of 28% contended that the megaloblastic anaemia in their patients was nutritional in origin and not due to ancylostorniasis because only 17 of their 100 patients with nutritional megaloblastic anaemia had hookworm infestation and these 17 patients responded to treatment with injections of crude liver extract without antecedant deworming. Layrisse et al presented evidence for decreased serum vitamin B12 levels and malabsorption of folic acid, while Gilles et al reported abnormal clearance of folic acid in six patients, although none of their patients had a megaloblastic bone marrow. Layrisse et al postulated that the malabsorption of folic acid may be on the basis that the highest concentration of hookworm exists at an intestinal level where folic acid is optimally absorbed. Serum folic acid or folic acid excretion was not tested in this series for lack of facilities.
Vitamin B12 absorption is generally reported to be within normal range in ancylostomiasis , ,  Sixteen patients in our series had low Coy B12 absorption. However, the worm infestation was probably not the cause of this impaired absorption since no improvement in absorption was observed after deworming. Further, some other cause for malabsorption was present in half of these patients. The anaemia in this series was also of iron deficiency type as reported by otheTs., , , ,, , , , , , , , , ,  Our studies confirm the reported finding of a significant relationship between worm load as determined by the egg count and the haemoglobin levels., , , , , , ,  Although haemoglobin levels are low, the bone marrow in hookworm infestation associated with iron deficiency anaemia shows a moderate erythroblastic hyperplasia, absent bone marrow haemosiderin and an increase in reticulocyte count., , , ,  Serum iron as observed in our series is invariably low and the unsaturated iron binding capacity is high., , , , , , 
It is generally accepted that blood and iron loss from the gastro-intestinal tract in ancylostomiasis is the cause of iron deficiency anaemia and this is confirmed by using  chromium labelled red cells., , , , , , , , , , , ,  Inspite of higher absorption, rapid disappearance of injected radioactive iron and rapid iron turn over,,  patients with hookworm infestation may still loose considerable quantities of iron via the feces. That the hookworm anaemia is mostly due to iron deficiency is further supported by the fact that it responds rapidly and dramatically to the administration of iron salts by mouth, and that it is possible to maintain the normality of blood values in individuals severely infected with ancylostomiasis by the administration of iron alone.,  This response to oral iron is independent of gastric acid levels. It may respond also to deworming with no change of diet, but over a longer time, provided the diet contains adequate amounts of iron.
Radio chromium tagged red cell survival time is generally reported to be decreased,, , , ,  the major site of destruction of the red blood cells probably being the spleen. The shortened survival is probably due to an intrinsic defect of the erythrocytes and not due to hemolysis and is not a cause but a result of severe iron deficiency anaemia, since deworming only partially corrects the shortened survival., , 
Vague epigastric pain, aggravated by meals and simulating duodenal ulcer has been described in ancylostomiasis,, , , ,  and it was once believed that hookworm may be an etiological factor in peptic (duodenal) ulceration. The gastric acid secretion has been generally reported normal or low., , , , , , , , , ,  Raju and Narielvala found a high basal acid output (BAO) in carefully `selected' subjects with ancylostomiasis, and attempted to link this increased BAO with a postulated excessive gastrin secretion secondary to duodenitis. However, this is unlikely since it remained high in their patients after deworming.
Radiological changes in the duodenum characteristic of ulcer have been reported by some.,  However, others, ,  did not find any increased incidence of duodenal ulcer in ancylostomiasis and our results support this finding. Other X-ray changes such as antiperistalsis, mucosal distortion, momentary dilatation and disordered motor function,  have been reported. Similar changes of mucosal distortion, momentary dilatation and disordered motor function were observed in our series. We did not observe any increased incidence of duodenal ulcer.
The question of malnutrition in hookworm disease is controversial; it is reported from India's while it is rare in reports from other areas., , , ,  It is difficult to separate the effects on blood values of dietary factors from those due to parasitic infection when both occur concurrently. Hookworm anaemia has been associated with a somewhat lower mean serum total protein level and a distinctly lower mean albumin. Gilles et al found that hypoalbuminaemia was always accompanied by depletion of the exchangeable albumin and that the patients who had become depleted were found to be turning over albumin at a less than the maximal rate. This is believed to be due to increased quantity of fecal nitrogen, rather than lack of absorption., 
Gilles et al found hepatic function normal in all patients except in the one who died with cirrhosis of the liver. The bromsulphthalein clearance was abnormal in their severely anaemic patients but in most cases it returned to normal on recovery. The liver functions and the liver biopsy studies in our series revealed no gross abnormality., 
A study of intestinal morphology and function in hookworm infection is of particular interest, since the parasites through the changes produced by its attachment to the mucosa could conceivably lead to impairment of the functions of digestion and absorption. The evidence for morphological changes has been conflicting; some workers have described diffuse histological changes,, , , , ,  while others have failed to find this., ,  In our series, only three patients had abnormal biopsy; two were suffering from tropical sprue while one had coeliac disease. This suggests that all other possible causes of malabsorption should be ruled out before the changes are ascribed to ancylostomiasis.
Carbohydrate absorption was assessed by means of glucose and lactose tolerant tests and by d-Xylose excretion. As a absorption test, glucose tolerance test not very useful since many norm, people may have a flat glucose tolerant test. Both glucose and lactose tolerant tests were normal in most of our patient; d-Xylose excretion test has also bee generally normal in most of the reports [Table 3]. Fat absorption in ancylostomiasis is also reported normal [Table 4] Our results confirm these findings. All seven patients with steatorrhoea in our series, as described above, had some other cause to account for this abnormality. Sheehy et al from Puerto Rico have reported histological changes and malabsorption in patients infected with ancylostomiasis while Layrisse et al from Venezuela have failed to find either malabsorption or histological changes. The differences regarding the reported morphological changes and the absorption studies in heavily infected subjects from Puerto Rico and Venezuela have been explained by Roche and Layrisse as due to frequent occurrence of sprue in Puerto Rico and its rarity in Venezuela. This is confirmed by the fact that the repeat tests after treatment in the series of Sheehy et a1 did not return to normal. It is also possible that some investigators reporting abnormalities have in fact studied patients with hookworm infection and pre-existing intestinal disease, especially where anaemia and the presence of hookworm ova in the stools have been the only criteria for selection. This is evident from our studies in patients with tropical sprue and coeliac disease in whom we had to repeat the studies not only after deworming, but also after a course of tetracycline, folic acid and gluten-free diet to come to a final diagnosis. Another likely factor for these divergent views is that there are true differences between patients, depending upon such factors as the duration of infection with ancylostomiasis, other parasitic infections such as giardiasis, site of biopsy, serum protein levels, etc. It is felt, therefore, that one should try to rule out all other possible causes of malabsorption by long term follow-up of patients with ancylostomiasis associated with malabsorption before one ascribes these changes to hookworm infestation.
The authors wish to thank the Dean, Seth G. S. Medical College, and K.E.M. Hospital, for permission to publish this paper.