Shaheen Shoaib, Shoaib Tauheed, Amtul Hafiz*

Department of Pathology, Dow Medical College, Karachi, *Department of Microbiology, BMSI, JPMC, Karachi

 Background: One of the important causes of childhood diarrhoea is cryptosporidium, a parasitic pathogen which is usually overlooked. This study was therefore designed to show the importance of modified acid fast stain in the diagnosis of cryptosporidium in childhood diarrhoea. Methods: Stool samples from 300 children with prolonged diarrhoea were examined by modified acid fast staining along with wet mounts in saline and iodine. Results: Out of 300 samples five (1.7%) were diagnosed as positive for cryptosporidium. Conclusion: Results indicate that Cryptospori-diosis exists as a cause of diarrhoeal illness in our society. It was also noted that modified acid fast staining of stool samples is an important, non-invasive and useful diagnostic technique in identification of cryptosporidium.

Key words: Cryptosporidium. Childhood diarrhea, Modified acid fast technique


Cryptosporidium is an enteric pathogen known to parasitise a wide range of animals and has a propensity to affect human beings.1

                Early human cases were reported in the United States in 1976 in an immunosuppressed adult and an immunologically competent child.2,3 An important aspect of cryptosporidiosis is its higher prevalence in young children with diarrhea.4,5

Molecular studies have shown that Cryptosporidium parvum is the major species involved in human infections.6 Most infections are acquired through direct contact with infected persons or through contaminated water or food.7,8

The infection is intracellular. The organism replicates and matures in enterocytes. The final product is the oocyst that passes out in the stools. The infection is highly transmissible.7,9 Medical history or physical examination are not sufficient for the diagnosis as diarrhoea is only a symptom caused by various pathogens through several mechanisms. However, cryptosporidium has been found to be significantly more prevalent in persistent diarrhea.5,10

Specific diagnosis depends on laboratory evaluation. Examination of stool for the characteristic oocysts by modified acid fast staining still remains an important diagnostic test.11 Diarrhoeal illness is a leading cause of morbidity and mortality in children. Although a number of enteric pathogens have been identified in recent years but many episodes of diarrhoea still remain undermined. Cryptosporidium has emerged as an important enteric pathogen10.

This study was designed to see the role of modified acid fast stain in the diagnosis of Cryptosporidium which is an unusual pathogen in children with prolonged diarrhoea.


This study involved 300 children presenting with chronic diarrhoea who came during the course of this study. Inclusion criteria was stool samples from children aged 1-14 years presenting with chronic diarrhoea in cross sectional manner. Cases presenting with acute diarrhoea or suggestive of bacterial or viral infection were excluded. None of the patients were receiving any immunosuppressive therapy or steroid treatment.

Samples were collected in clean, dry, leak-proof glass containers. Each sample was examined for gross appearance. Presence of worm segments, mucus or blood was noted.

For microscopic examination wet preparations in saline and iodine were prepared and examined for ova or cysts.

Smears for modified acid fast staining were prepared by taking a pea sized material from the stool sample by a glass rod and making an even, thin smear on clean glass slide. It was left to dry and fixed with 70% alcohol.

After fixation smear was flooded with cold carbol fuchsin and left for ten minutes. Smear was decolourized with 1% HCl alcohol until colour ceased to flow. Smear was rinsed with water and counter stained with methylene blue for 30 seconds. The preparation was rinsed again with plain water, dried and examined under oil immersion objective. Oocysts of Cryptosporidium appear as bright rose pink spherules against light blue background. The modified acid fast technique is a sensitive diagnostic method as recommended by WHO.11


Out of 300 cases five were found to be positive for Cryptosporidiosis as shown in the table. History of clinical illness of all cases revealed prolonged, persistent diarrhoea. Five cases that were positive for Cryptosporidium did not give any history of nausea, vomiting or loss of appetite associated with diarrhoea. Stools were frequent and watery.

On gross examination of the five positive cases presence of worm segments or blood was not observed. In two cases mucus was present. In the remaining cases presence of blood and mucus was frequent.

Examination of stool samples by wet mounts failed to reveal oocysts of Cryptosporidium which were found positive on modified acid fast staining in five cases. Only one case positive for Cryptosporidium was also positive for Giardia cyst showing mixed infection.

Table-1: Clinical and Laboratory Findings in Cases of Cryptosporidiosis



Age & sex

Duration of


Lab. Finding


Wet preparation


acid fast stain


7  yrs






Giardia cysts +ve

Oocysts +ve


8 yrs





 water mucus +ve

No cysts or ova observed

- do -


11 yrs



 months `


No cysts or ova

-do -


8 yrs





- do -



10 yrs




Loose with mucus




The results of this study highlight the fact that unusual pathogens like Cryptosporidium should not be overlooked while treating cases with diarrhoea. Although the frequency was found to be 1.7% but it shows the existence of this unusual pathogen in our population.

Cryptosporidium has emerged as an important cause of diarrhoea world wide. This parasite has been reported not only from Western countries but also from Nigeria, Sudan, Srilanka, Bangladesh and India with a strong possibility for it to be present in Pakistan also.10

Cryptosporidium is recognized as a major cause of waterborne diarrhoeal illness and as a pathogen with long term effects on childhood growth and development.12 The most common manifestation of this parasite was prolonged diarrhoea13 as also seen in the aforesaid study.

In a study carried out on 63 Pakistani travellers to UK, oocysts of Cryptosporidium were found in 13 travellers presenting with diarrhea.14 Another study was carried out on water samples in Lahore. Out of 70 water samples 9% were found to be positive for oocysts of Cryptosporidium.15 In a study carried out on duodenal biopsies in Karachi, four cases were reported to be positive for Cryptosporidium. These cases presented with chronic diarrhoea and stool examination by routine wet mounts did not reveal any significant finding.16

Cases of prolonged diarrhoea are usually not referred by clinicians for laboratory diagnosis of Cryptosporidosis as most of the clinicians especially in Pakistan are still unaware of Cryptosporidiosis in immunocompetent children and they are not sure of the symptoms caused by it.

Cryptosporidium is an important etiological agent of childhood diarrhoea and should be identified in routine parasitologic tests of diarrhoeal stool samples.17 Recognition and diagnosis of this pathogen is of utmost importance for the prompt treatment of such cases. Modified acid fast staining is a sensitive method that is economical and easily applicable as compared to invasive procedures like duodenal biopsies. This method can improve detection of common source out breaks as well as in constructing timely public health interventions.


Cases of cryptosporidium in childhood diarrhoea were diagnosed by modified acid fast staining technique. These positive cases were missed on wet mount. Modified acid fast staining was easily applicable and economical mode of diagnosis. Cases with persistent diarrhoea should be referred for diagnosis of Cryptosporidium


1.        Esteban JG, Aguirre C, Flores A, Strauss W, Angles Rene, MasComa S. High cryptosporidium prevalence in healthy Aymara children. Am J Trop Med Hyg 1998; 58:50-55.

2.        Nime FA, Burck D, Page DL, Holscher MA, Yardley JH. Acute enterocolitis in a human being infected with protozoan cryptosporidium. Gastroenterology 1976; 70:592-6.

3.        Meisel JL, Perera DR, Meligro C, Rubin CE. Over whelming watery diarrhoea associated with cryptosporidium in an immunosuppressed patient. Gastroenterology 1976;70:1156-60.

4.        Xian ZUS, Fen LIJ, Barrett LJ, Fayer R, Shu SY, McAuliffe JF et al. Seroepidemiologic study of cryptosporidium infection in children from rural communities of Anhui, China. Am J Trop Med Hyg 1994; 51:1-10.

5.        Molbak K, Wested N, Hojlying N, Scheutz F, Gottschau A, Aaby P et al. The etiology of early childhood diarrhoea A community based study from Guinea Bissau. J Infect Dis 1994;169:581-7.

6.        Checkley W, Epstein LD, Gilman RH, Black RE, Cabrera L, Sterling CR. Effects of cryptosporidium parvum infection in Peruvian children. Am J Epidemiol 1998; 148:497-506.

7.        Goodgame RW. Understanding intestinal spore forming protozoa. Ann Intern Med 1996; 124:429-41.

8.        Mosier DA, Oberst RD. Cryptosporidiosis. A global challenge. Annals New York Acad Sci 2000; 916:102-11.

9.        Newman KD, Zu SX, Wuhib T, Lima AA, Guerrant RL, Sears CL. House hold epidemiology of cryptosporidium parvum infection in an urban community in northern Brazil. Ann Intern Med 1994; 120:518-19.

10.     Baqai R. Cryptosporidium in diarrhoeal disease (editorial). JPMA 1990; 40:174-5.

11.     WHO Basic Laboratory methods in Medical Parasitology. Geneva WHO 1991.

12.     Kosek M, Alcantara C, Lima AA, Guerrant RL. Cryptosporidiosis. An Update. Lancet Infect Dis 2001; 4:262-9.

13.     Morin CA, Roberts CL, Patricia A, Addis DG, James LH. What do physicians know about cryptosporidiosis? Arch Intern Med 1997; 157:1017-22.

14.     Flegg PJ. Cryptosporidium in travelers from Pakistan. Trans R Soc Trop Med Hyg 1987; 81: 171-81.

15.     Memon MM. Cryptosporidium A waterborne pathogen. Pak J Med Res 1997; 36:47-9.

16.     Abbas Z, Sheikh H, Jafri SMW, Khan AH, Protracted cryptosporidiosis in adults diagnosed by distal duodenal biopsies. Specialist 1994;10:273-5.

17.     Pereira MD, Atwill ER, Barbosa AP, Silva A, Garcia ZMT. Intra-familial and extra-familial risk factors associated with Cryptosporidium parvum infection among children hospitalized for diarrhoea in Goiania, Goias, Brazil. Am J Trop Med Hyg 2002; 66:787-93.

Address of Correspondence:

Dr.Shaheen Shoaib, F-3, Hasan Center, Gulshan-E- Iqbal, Block-16,Karachi.