Cryptosporidium is a major enteric pathogen of patients with acquired immunodeficiency syndrome (AIDS), with infection rates of 8–48% reported among African AIDS patients with diarrhea.1–4 Studies of patients with cryptosporidiosis indicate that a majority experience chronic diarrhea, while less than 15% have transient diarrhea or are asymptomatic.5,6 However, this asymptomatic rate may be an underestimate because relatively few AIDS patients without diarrhea have been tested for Cryptosporidium infection in such studies. Additionally, the relative clinical impact of the two major Cryptosporidium species that infect humans, C. hominis (also referred to as genotype 1) and C. parvum (also referred to as genotype 2), is poorly defined. A predominance of C. hominis infection has been observed in HIV-associated diarrhea patients in Peru7 and Thailand,8 C. parvum has been prevalent in Europe,9,10 while genotype data from Africa have been limited to only a few cases.11–13
We performed a cross-sectional study of HIV-suspected inpatients in the Kilimanjaro region of Tanzania at the Kilimanjaro Christian Medical Centre, the Mawenzi Government Hospital, and the Kibong’oto National Tuberculosis Hospital from July to October 2002. Informed consent was obtained from all participants and the University of Virginia Human Investigation Committee and the Kilimanjaro Christian Medical Centre Ethics Committee reviewed and approved the project. The hospitals’ standard HIV testing and counseling procedures of the hospitals were maintained and no patient was tested for HIV for the purpose of entry into the study. Patients with previously documented or newly suspected HIV infection were eligible. Enrolled patients were asked if they were experiencing increased stool frequency (≥ 3 liquid stools/day), the duration if present, and a single blood and stool sample was obtained. This study took place before the era of widespread availability of anti-retroviral therapy in this region.
Stool specimens were tested for Cryptosporidium infection by immunofluorescence (IF) microscopy (MeriFluor™ Cryptosporidium/Giardia; Meridian Bioscience, Inc., Cincinnati, OH) and by a nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) according to the protocol of Sturbaum and others.14 DNA was isolated from aliquots of frozen stool using the QiAMP DNA stool mini kit (Qiagen, Valencia, CA) after two washes in sterile phosphate-buffered saline (PBS) and six cycles of freeze-thaw. For each PCR, water (no DNA) and positive control DNA from C. parvum oocysts were used to confirm absence of contamination and successful amplification, respectively. The PCR assay exhibited a sensitivity of 94% and a specificity of 96% versus the reference IF assay15 and detected five IF-negative infections. All positive PCR results were repeated for verification and a positive PCR or IF result was considered valid. In addition to Giardia, which was tested by the IF assay, other stool pathogens were evaluated as follows: an enzyme-linked immunosorbent assay (ELISA) for Entamoeba histolytica (E. histolytica II ELISA assay; Techlab, Blacksburg, VA), culture for enteric bacteria using Selenite F broth and deoxycholate citrate and MacConkey agars, and microscopy for ova and parasites on formalin-ether sediments.
Two hundred forty-five patients were enrolled and complete data were obtained from 156, of which 127 were HIV-1/2 positive by ELISA (Vironostika Uniform HIV plus O; Organon Teknika B.V., Boxtel, The Netherlands or GenScreen HIV 1/2, Bio-Rad Laboratories, Marnes la Coquette, France). Quantification of CD4 T cells was performed on EDTA-containing blood within four hours of collection using the Coulter Manual CD4 Count kit16 (Beckman Coulter, Hialeah, FL). Cryptosporidium infection was a marker for HIV infection in that 22 of 127 HIV-positive patients were infected versus 0 of 29 HIV-suspected but HIV-negative patients (P = 0.01). Clinical features of the HIV-positive population are summarized in Table 1. At least 53% (67 of 127) of patients met the AIDS surveillance case definition via a CD4 cell count < 200/μL.17 Diarrheal symptoms (≥ 3 liquid stools/day) were reported by 48% of the patients and had been present for an average of duration of one month.
Upon examining the relationships between Cryptosporidium infection, diarrheal symptoms, and CD4 cell count (Table 2), we found that Cryptosporidium-infected patients had lower CD4 cell counts than Cryptosporidium-negative patients (median = 124/μL versus 212/μL; P < 0.04). Similar to the findings of others,1,2 we found that diarrheal symptoms also correlated with a low CD4 cell count (median = 139/μL versus 226/μL; P = 0.02). We were surprised to find that asymptomatic infection with Cryptosporidium was common (11 of 66 versus the 11 of 61 symptomatic Cryptosporidium infection rate; P not significant), particularly given the low CD4 cell counts (median = 128/μL) of these patients. Indeed, this low CD4 cell count was a distinguishing feature of Cryptosporidium-infected asymptomatic patients (median = 128/μL versus 336/μL for Cryptosporidium-negative asymptomatic patients; P = 0.02), indicating that Cryptosporidium infection was a marker for advanced lymphopenia independent of diarrhea. Cryptosporidium loads estimated by the method of Goodgame and others18 were variable and did not significantly differ between patients with symptoms and those without symptoms (mean ± SD/mL of sediment = 15,000 ± 43,000 versus 5,600 ± 12,000; P not significant). Of note, only 1 of 11 Cryptosporidium-infected patients with diarrheal symptoms was co-infected with other enteric pathogens as tested (one with a Giardia co-infection and none with E. histolytica, Strongyloides, Salmonella, or Shigella co-infections).
Since Cryptosporidium was an important marker in this population for advanced CD4 lymphopenia, we sought to determine whether this risk was influenced by Cryptosporidium species. Of the 21 stool specimens that were PCR-positive for Cryptosporidium (one specimen was PCR−/IF+), RFLP analysis showed that 15 were C. hominis and 6 were C. parvum (Table 3). There was no association between species and diarrheal symptoms. However, the duration of symptoms in C. hominis-infected patients was longer than that of C. parvum-infected patients, and the rate of asymptomatic C. hominis infection was higher (14% versus 2% for C. parvum; P = 0.02). Interestingly, the median CD4 cell count of C. hominis-infected patients was lower than that of C. parvum-infected patients, and this was true for both symptomatic and asymptomatic groups. Again, there was no statistical difference in the average parasite load between C. hominis and C. parvum (mean ± SD/mL of sediment = 10,000 ± 37,000 versus 5,000 ± 8,000; P not significant).
The most important new findings in this study are the high rate of asymptomatic C. hominis infection in this population and the apparent preferential association of C. hominis infection with a low CD4 cell count. The 17% rate of asymptomatic Cryptosporidium carriage in this study is higher than the 1–5% asymptomatic rate previously reported in AIDS inpatients from many developing world sites.2,19,20 The high rate was not due to our use of a PCR-based assay (since 9 of 10 asymptomatic patients were also IF positive) and is surprising given the low CD4 cell count of this asymptomatic group (128/μL), since other studies have suggested that chronic symptoms are the norm in this CD4 range.5 We believe there may be an underappreciation of asymptomatic Cryptosporidium infection, at least in this region of Africa, given that most clinical data on cryptosporidiosis derives from series of patients with diarrhea and not asymptomatic controls.3,21–23 It may be relevant that community-based studies from Bolivia24 and Korea25 have shown asymptomatic Cryptosporidium carriage rates of 8–32% in large numbers of healthy individuals. Another feature of our patient population was the low Cryptosporidium oocyst loads relative to what others have observed,18 which could be contributing to the relative lack of symptoms, or simply reflect biological variability or laboratory differences.
Important limitations of our study are the small sample size, the inherent risk of recall bias when evaluating diarrheal symptoms, and the lack endoscopy or serial stool examination. We are interested in determining if these asymptomatic Cryptosporidium-infected patients were transiently or chronically infected, and whether they were forever symptomatic, went on to become symptomatic in the future, or had symptoms in the remote past. One could speculate that the preferential detection of C. hominis in patients with low CD4 cell count is due to delayed clearance of the organism, perhaps from symptomatic infections acquired during the previous wet season when transmission and new infection are common.26 Highly powered, prospective longitudinal studies are needed to confirm our findings of asymptomatic C. hominis carriage with low CD4 cell count, and to determine whether such a relationship is cause versus effect.
Clinical features of the human immunodeficiency virus–infected study population (n = 127) Kilimanjaro, Tanzania
| Feature | |
|---|---|
| Age, years (median, range) | 35 (18–65) |
| Sex (M:F) | 47:80 |
| Cryptosporidium infection | 22 (17%) |
| CD4 cell count (median/μL) | 179 |
| Symptoms of ≥ 3 liquid stools/day | 61 (48%); median duration = 1 month |
Relationships between Cryptosporidium infection, symptoms, and CD4 cell count*
| ≥ 3 liquid stools/day | Asymptomatic | Total | |
|---|---|---|---|
| * CD4 cell values are reported as median/μL; the value was not available for one Cryptosporidium-positive asymptomatic patient. Statistical analyses were based on the Mann-Whitney test. | |||
| † P < 0.04, comparing the median CD4 cell count of Cryptosporidium-positive vs. negative patients. | |||
| ‡ P < 0.04, comparing the median CD4 cell count of Cryptosporidium-negative asymptomatic patients versus positive/asymptomatic, negative/symptomatic, or positive/symptomatic groups. | |||
| § P = 0.02, comparing the median CD4 cell count of patients with vs. without symptoms. | |||
| Cryptosporidium positive | 11 (CD4 = 124) | 11 (CD4 = 128) | 22 (CD4 = 124†) |
| Cryptosporidium negative | 50 (CD4 = 150) | 55 (CD4 = 336‡) | 124 (CD4 = 212†) |
| Total | 61 (CD4 = 139§) | 66 (CD4 = 226§) | |
Comparison of Cryptosporidium hominis versus C. parvum infection*
| C. hominis | C. parvum | |
|---|---|---|
| * CD4 cell counts/μL and duration of symptoms are reported as median values; the CD4 cell count was not available for one asymptomatic C. hominis-infected patient. | ||
| † P = 0.009 comparing duration of symptoms of C. hominis vs. C. parvum-infected patients (by Mann-Whitney test). | ||
| ‡ P = 0.02 comparing rates of asymptomatic infection with C. hominis vs. C. parvum (by Fischer’s exact test). | ||
| § P < 0.003 comparing CD4 cell counts of C. hominis vs. C. parvum-infected patients (by Mann-Whitney test). | ||
| Total | 15 (71%) | 6 (29%) |
| Association with symptoms | 6/61 (10%) | 5/61 (8%) |
| Duration of symptoms (months) | 4† | 0.3† |
| Association with asymptomatic infection | 9/66‡ (14%) | 1/66‡ (2%) |
| CD4 cell count | 55§ | 256§ |
Address correspondence to Eric R. Houpt, Division of Infectious Diseases and International Health, University of Virginia, PO Box 801340, MR4 Building Room 2144, Charlottesville, VA 22908-1340. E-mail: erh6k@virginia.edu
Authors’ addresses: Eric R. Houpt, Oluma Y. Bushen, Anita Kohli, Amon Asgharpour, Cherie T. Ng, and Richard L. Guerrant, Division of Infectious Diseases and International Health, University of Virginia, PO Box 801340, MR4 Building Room 2144, Charlottesville, VA 22908-1340, Telephone: 434-243-9326, Fax: 434-924-0075, E-mails: erh6k@virginia.edu, ob3d@vurginia.edu, ak7t@virginia.edu, aa3z@virginia.edu, ctn8d@virginia.edu, and rlg9a@virginia.edu. Noel E. Sam, Venance Maro, Sendui Ole-Nguyaine, and John F. Shao, Kilimanjaro Christian Medical Centre, Moshi, Tanzania, E-mails: nsam@kcmc.ac.tz, venmaro@yahoo.co.uk, solengudr@yahoo.com, and jshao@kcmc.ac.tz. David P. Calfee, Division of Infectious Diseases, Mount Sinai School of Medicine, New York, NY 10029, E-mail: David.Calfee@msnyuhealth.org.
Acknowledgments: We thank Edward Mushi, Sr., Ruwaichi Uiso, and the other nurses involved in this study; Stanislaus Siriwa, Richard Tarimo, and Eline Ngomuo and the other clinical laboratory technicians; and all the patients who participated in this work. We also thank Dr. William Petri (University of Virginia) for helpful discussions and David Lyerly (Techlab, Inc.) for the E. histolytica II ELISA kits.
Financial support: This study was supported by the National Institutes of Health (grant U19 AI056872-01) and the Virginia Commonwealth Technology Research Fund.
Disclosure: None of the authors has a commercial or other association that might pose a conflict of interest.
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