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Year : 2018  |  Volume : 36  |  Issue : 4  |  Page : 508--512

A road less travelled: Clinical comparison of HIV seropositive and seronegative patients with cystoisosporiasis – An 11-year experience from a tertiary care centre in Northern India

Ujjala Ghoshal1, Vidhi Jain1, Nidhi Tejan1, Sonali Khanduja Kalra1, Prabhat Ranjan1, Richa Sinha1, Dinesh Gangwar1, Uday C Ghoshal2,  
1 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Correspondence Address:
Prof. Ujjala Ghoshal
Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow - 226 014, Uttar Pradesh
India

Abstract

Background: Cystoisospora is a well-known opportunistic enteric parasite among human immunodeficiency virus (HIV) seropositive patients but there is a paucity of data among HIV negative patients. This study investigated Cystosporiasis on both HIV positive and negative patients, with or without diarrhea, presenting to a tertiary care and super specialty center of northern India. Methodology: Oocysts of Cystoisospora were detected on light microscopy, by modified Kinyoun staining of stool specimens, over an 11-year study period. Results: Of the 10,233 stool specimens evaluated, Cystoisospora was detected in 64 patients, 37 (57.81%) of whom were HIV positive. Year-wise analysis showed an overall declining trend of cystoisosporiasis. Maximum cases were detected in May and June in HIV positive patients and February and September among HIV negative patients. Among HIV positive patients, the mean CD4 count was 152.04 ± 81.12cells/μL, mean absolute eosinophil count (AEC) was 229.16 ± 175.62 cells/μL and 12.5% patients had mild eosinophilia. Tuberculosis was the most common co-morbidity. Dual infections of Cystoisospora with Cryptosporidium and Giardia were also seen. Among HIV negative patients, eight had primary autoimmune disorders, seven were solid organ transplant recipients and the rest had chronic bowel diseases. The mean AEC was 485.47 ± 414.88 cells/μL, with 14.81% patients showing mild and 11.11% showing marked eosinophilia. Dual infection with Giardia was seen. Recurrent cystoisosporiasis was noted, despite cotrimoxazole treatment in a single case. Conclusion: The epidemiology of cystoisosporiasis differs between HIV seropositive and seronegative patients in terms of year-wise and month-wise trends, co-infections and most importantly, AECs.

How to cite this article:
Ghoshal U, Jain V, Tejan N, Kalra SK, Ranjan P, Sinha R, Gangwar D, Ghoshal UC. A road less travelled: Clinical comparison of HIV seropositive and seronegative patients with cystoisosporiasis – An 11-year experience from a tertiary care centre in Northern India.Indian J Med Microbiol 2018;36:508-512

How to cite this URL:
Ghoshal U, Jain V, Tejan N, Kalra SK, Ranjan P, Sinha R, Gangwar D, Ghoshal UC. A road less travelled: Clinical comparison of HIV seropositive and seronegative patients with cystoisosporiasis – An 11-year experience from a tertiary care centre in Northern India. Indian J Med Microbiol [serial online] 2018 [cited 2019 Nov 16 ];36:508-512
Available from: http://www.ijmm.org/text.asp?2018/36/4/508/254404

Full Text

 Introduction



Cystoisospora belli is a well-known opportunistic parasite. It was first discovered by Virchow in 1860 and earlier called Isospora belli by Wenyon in 1923.[1] In immunocompetent patients, it causes a mild self-limiting diarrhoea and has been implicated in traveller's diarrhoea.[2] However, in immunocompromised patients, it can present with persistent or chronic non-bloody diarrhoea, indistinguishable from that caused by other opportunistic intestinal pathogens such as Cryptosporidium, Cyclospora and Microsporidia. Opportunistic infections remain the hallmark of human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS). Cystoisosporiasis is an AIDS defining illness.

As per Global AIDS Response Progress Reporting 2016, a colossal 36.7 million people were suffering from HIV/AIDS worldwide, of which 2.1 million resided in India.[3] In a study of more than 16,000 HIV-infected patients between 1985 and 1992 in Los Angeles County, USA, the prevalence of Cystoisospora infections was highest in foreign-born patients, particularly those from Mexico and El Salvador, and among persons of Hispanic ethnicity.[4] Among Asian countries, India reports the maximum cases of this opportunistic parasite. In India, the prevalence of cystoisosporiasis ranges from 2.5% to 26.1% in HIV-positive patients.[5],[6],[7],[8],[9] However, there is a paucity of data on the occurrence of cystoisosporiasis among HIV-negative patients.

Therefore, we conducted this prospective observational study to compare the occurrence of cystoisosporiasis among HIV seropositive and seronegative patients, presenting to a tertiary care and referral centre in northern India over 11 years, with the following objectives: To study:

Demographic details of the patients with CystoisosporaMonthly and yearly trends in the detection of CystoisosporaClinical presentation, co-infections and association with highly active antiretroviral therapy (HAART) and anti-tubercular therapy (ATT)Laboratory parameters such as CD4 counts and absolute eosinophil counts (AEC).

 Patients and Methods



This study was carried out prospectively on immunocompromised patients, both HIV positive and negative, including patients with haematologic malignancies and solid organ transplant, during 11 years from 1 January 2007, to 31 December 2017. Stool specimens from both HIV positive and negative patients with or without diarrhoea, presenting to a tertiary care centre, were analyzed for the presence of opportunistic pathogens, including Cystoisospora. Stool specimens from 200 healthy controls were also included in this study.

Stool specimens were collected in clean, wide-mouthed, screw-capped disposable containers and processed within 1 h, for microscopy by direct wet mount and the modified Kinyoun technique.

Direct wet mount

Both iodine and normal saline mounts were prepared as per standard method. With the help of an applicator stick, one part of the stool sample was emulsified in a drop of saline on a clean, dry slide and in a drop of Lugol's iodine. These were covered with coverslips and observed under low and high power of light microscope for the detection of oocysts of Cystoisospora.

Formol-ether concentration technique

It was performed by the standard method, as previously reported.[10] The supernatant was discarded and sediment collected for the modified Kinyoun technique.

Modified Kinyoun technique

It was performed with a few modifications, as previously reported by us.[10] The smear was scanned on a 10× eyepiece and oil immersion objective 100× of the light microscope, with an effective magnification of 1000×. Oocysts of Cystoisospora passed in stool were seen as ellipsoidal reddish-pink structures measuring approximately 20–30 μm in length and 10–19 μm in breadth; depending on maturity, they were unsporulated, sporulated with only one sporoblast or mature with two sporoblasts.

Demographic and clinical details of the patients were recorded on a predesigned proforma. Statistical analysis was performed on Statistical Package of social sciences (SPSS) version 15 (SPSS Inc., Chicago, IL, USA).

 Results



Over the 11-year study, the parasitology laboratory of our institute received 10,233 stool specimens for detection of opportunistic parasitic pathogens in immunocompromised patients. A total of 64 patients were found to excrete oocysts of Cystoisospora, of which 37/64 (57.81%) were seropositive for HIV. The majority (93.75%) presented with diarrhoea, while four (6.25%) patients did not. AEC was available for 59 patients. Mean AEC was 312.41 ± 362.21 cells/μL. Cystoisospora was were seen among immunocompetent controls.

Demographic details of patients

The mean age of patients was 35.63 ± 11 years (ranged 7–60 years). All were adults except one, who was 7-year-old. Maximum cases i.e., 30/64 (46.88%) belonged to the age group of 30–40 years with a male to female ratio of 2.56. Most patients 42/64 (65.63%) were outpatients. Among inpatients, two HIV positive and two HIV negative eventually died due to bacterial sepsis.

Cystoisosporiasis among HIV-positive patients

Of 64 patients, who tested positive for oocysts of Cystoisospora in stool, 37 (57.81%) were HIV positive. Most 8/37 (21.62%) cases were seen in the year 2007, followed by 7/37 (18.92%) in 2008, with maximum detection between May and June. The mean age of patients was 34.84 ± 9.93 years (range 7–65 years). Male to female ratio was 4.29. Most 34/37 (91.89%) patients presented with diarrhoea.

All 37/64 patients were positive for HIV-1 and none for HIV-2. Tuberculosis was the most common co-morbidity and 13/37 (35.14%) patients were already receiving ATT at the time of diagnosis of cystoisosporiasis. CD4 counts could be done in 27/37 (72.97%) patients and ranged between 09 and 309 cells/μL with a mean of 152.04 ± 81.12 cells/μL. The average haemoglobin level was 10.82 ± 2.04 g/dl. AEC was available for 32 patients and mean AEC was 229.16 ± 175.62 cells/μL.

Cystoisosporiasis among HIV-negative patients

Twenty seven of 64 (42.19%) patients positive for Cystoisospora, were HIV negative. Of them, 7 (25.93%) had chronic diarrhoea, 8/27 (29.63%) had autoimmune disorders, 6/27 (22.22%) were renal transplant recipients, 3/27 had ulcerative colitis and one each Crohn's disease, liver transplantation and anaplastic meningioma (on radiotherapy). All patients presented with diarrhoea, except one.

Most (22.22%) patients were seen in 2006, followed by 4 (14.81%) cases each in 2004 and 2015 with most frequent parasite isolation between February and September. The mean age of patients was 36.70 ± 12.25 years (range17–60 years). Male to female ratio was 3.0. The average haemoglobin level was 10.81 ± 2.62 g/dl. AEC was available for all 27 patients and mean AEC was 485.47 ± 414.88 cells/μL.

 Discussion



Over the 11-year study, 10,233 stool specimens from both HIV positive and negative patients, with or without diarrhoea, yielded 64 cases (0.67%) positive for oocysts of Cystoisospora. This observation resonates with 0.7% reported by a study from Burkina Faso, Africa [11] but is much lower than the range (2%–6%) reported by other studies from India.[12],[13],[14] The disparity could be due to geographical factors, smaller sample size in these studies (200–1700 patients), shorter duration (3 months–5 years), clinical presentation with diarrhoea as an inclusion factor and higher preponderance of HIV-positive patients. Only 37/64 (57.81%) patients with cystoisosporiasis in our study were HIV-positive patients.

Patient demographic characteristics

Most patients were in the 30–40 years of age group, which might have been biased by the absence of paediatrics outpatient department in our institute. The male preponderance of cases matches most studies worldwide.

Trends in detection of cystoisosporiasis

On year-wise analysis, an overall declining trend of cystoisosporiasis was noticed from 2007 to 2014 as depicted in [Figure 1]. This finding is in contrast to the rising trend reported from southern India.[6],[15] The discordance can be explained by differences in the study population and geographical location. Those studies were conducted exclusively on HIV-positive patients, while we studied a mixed population. A declining trend was seen even among HIV-positive cases, probably due to the prophylactic administration of trimethoprim and sulfamethoxazole (TMP-SMX).{Figure 1}

Case detection among HIV-negative patients rose after 2011, probably due to the increasing awareness among physicians regarding cystoisosporiasis associated diarrhoea in non-HIV patients.

Month-wise detection, as depicted in [Figure 2], peaked during summer months of May (12.82%) and June (15.38%) among HIV-positive patients, while, HIV-negative cases presented most often during the spring months of September (20%) and February (16.67%). Interestingly, no case of Cystoisospora was detected among HIV-positive patients in February during these 11 years. Month-wise analysis from a previous study from northern India has shown May–August to show the maximum (63.41%) case detection over a 5-year study.[13]{Figure 2}

Persistence/recurrence of cystoisosporiasis is uncommon after TMP-SMX therapy. It has been reported from HIV-positive patients in Africa [16] and from a single HIV-negative immunocompetent patient from Thailand,[17] emphasizing the need for follow-up stool examinations in patients with long-term immunosuppression. A single HIV-negative renal transplant recipient presented with cystoisosporiasis in our study, 2 years after receiving a full course of treatment.

Clinical presentation, co-infections and association with highly active antiretroviral therapy and anti-tubercular therapy

Majority (60/64, 93.75%) of cystoisosporiasis cases presented with diarrhoea. Most patients were diagnosed and treated on an outpatient basis. Only 22/64 (34.38%) had symptoms, which were severe enough to warrant hospital admission. Four such patients eventually died due to bacterial sepsis, unrelated to cystoisosporiasis.

Most cases of cystoisosporiasis were mono-parasitic infections. Two HIV-positive patients presented with dual infection with both Cystoisospora and Cryptosporidium, similar to some case reports.[12],[18],[19] Two HIV negative, renal transplant recipients and one HIV seropositive patient suffered from coinfection with trophozoites of Giardia. The same has been previously reported by us among patients with microsporidiosis [10] and cryptosporidiosis (unpublished data).

Tuberculosis was the most common co-morbidity, and it was associated with 35.14% HIV-positive patients with cystoisosporiasis in our study, a finding corroborated by other studies from India.[15],[20] Other co-morbidities were herpes zoster and oral thrush, similar to previous reports.[12],[15]

In HIV-positive patients, cystoisosporiasis is an AIDS-defining condition and administration of HAART reduces the overall incidence of opportunistic parasites.[21] For 27 of our patients, cystoisosporiasis was the first clue for the suspicion of HIV infection, and they were HAART naïve. Five patients were on regular HAART while in five other patients HAART had been withheld due to side effects at the time of diagnosis of cystoisosporiasis.

There have been very few studies that have evaluated cystoisosporiasis among HIV-negative immunocompromised patients. We found 21/27 (77.78%) cases among HIV-negative patients. Of these, 15/27 (55.56%) patients had definitive diagnosis of immunosuppression, 8 (29.63%) patients had autoimmune disorders such as systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, X-linked hyper immunoglobulin G syndrome and chronic variable immunodeficiency. Cystoisosporiasis has previously been reported among patients with hypogammaglobulinemia,[22] Evans syndrome [23] and pemphigus vulgaris.[24] Seven patients suffered from chronic diarrhoeas due to ulcerative colitis, Crohn's disease and tropical sprue. To the best of our knowledge, only a single case of cystoisosporiasis has been reported in ulcerative colitis till date.[25]

The rest 12/27 (22.22%) were solid organ transplant recipients (11 renal and 1 liver transplant), despite cyclosporine therapy, which has strong parasiticidal effects. Three routes of infection have been proposed: Transmission with the graft, de novo infection and reactivation of latent infection as a consequence of immunosuppression.[26] Cystoisosporiasis among renal and hepatic transplant recipients has been previously reported from India.[26],[27]

Laboratory parameters

The occurrence of cystoisosporiasis strongly correlated with low CD4 counts (<300 cells/μl) in HIV seropositive patients in our study, a finding corroborated by multiple studies worldwide.[4],[5],[15],[20]

Parasitic diseases are classically associated with peripheral eosinophilia i.e., >5% eosinophils on differential leucocyte count. AEC, a better indicator of eosinophilia, is calculated by multiplying eosinophil percentage with total leucocyte counts (TLC) and has a normal range of 30–350/μL. AEC values were available for 59/64 patients. Mean AEC was 362.21 ± 312.41/μL. The mean AEC of HIV-negative patients (485.47 ± 414.88/μL) was almost double that of HIV-seropositive patients (229.16 ± 175.62/μL), presumably due to the higher overall TLC seen in HIV-negative patients. To the best of our knowledge, no previous study has evaluated AEC differences between HIV seropositive and negative patients.

Mild eosinophilia (AEC >500/μL) was seen in 4/32 (12.5%) of HIV seropositive and 4/27 (14.81%) of HIV seronegative patients in our study. The same has been previously reported in 50% of cases of cystosiosporiasis in two studies [28],[29] and a few isolated case reports.[30],[31],[32],[33]

Marked eosinophilia (AEC >1500/μL; range 1138–2193/μL) was present in 3/27 (11.11%) HIV-negative patients in our study but was absent from HIV seropositive patient group. Marked eosinophilia has never been reported in HIV-negative patients with cystoisosopirasis before, to the best of our knowledge.

AEC calculation might be an important adjunctive marker and should be included in the diagnostic algorithm while evaluating HIV-negative patients for cystoisosporiasis. However, lower AEC values in HIV-positive patients might be misleading and should not be the basis of ruling out opportunistic parasitosis in this group.

To summarize, this is the largest study on cystoisosporiasis from India, evaluating both HIV seropositive and negative patients, spanning 11 years. Males in 30–40 years of age group were most frequently affected. There was an overall declining trend in the detection of the parasite. Among HIV-positive patients, cystoisosporiasis was most frequently associated with HAART-naïve adults with CD4 counts <300 cells/μl. Tuberculosis was the commonest co-morbidity. The detection of this parasite should spark a thorough search for underlying immunodeficiency. Follow-up stool examinations to diagnose recurrence should be carried out in all patients, even after a full course of TMP-SMX treatment.

Among HIV-negative patients, cystoisosporiasis was associated with primary immunodeficiency disorders and immunosuppression secondary to organ transplant. Mean AEC of HIV seronegative patients was almost double that of HIV-positive patients, with 11.11% cases showing marked eosinophilia, a novel finding reported by our study. We suggest AEC as a useful adjunctive marker among HIV seronegative patients undergoing evaluation for cystoisosporiasis.

Ethical clearance

The Review Board and Ethical Committee of SGPGI, Lucknow, India, have approved the study (A-29: PGI/EP/EC/37/20/3/2007).

Acknowledgement

We gratefully acknowledge all the participants for agreeing to participate in the study. Support of all the staff members of our laboratory is also duly acknowledged.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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