Daniel Segovia Vargas

Differences in clinical manifestations among Cryptosporidium species and subtypes in HIV-infected persons

We performed a cross-sectional study to determine the epidemiology of Cryptosporidium in human immunodeficiency virus (HIV)-infected persons at 3 diagnostic levels: microscopy, genotypes of Cryptosporidium, and subtype families of C. hominis and C. parvum. The study enrolled 2,490 HIV-infected persons in Lima, Peru, and 230 were microscopy positive for Cryptosporidium infection. Specimens from 193 participants were available for genotyping. They had C. hominis (141 persons), C. parvum (22 persons), C. meleagridis (17 persons), C. canis (6 persons), C. felis (6 persons), and C. suis (1 person) infection. Although microscopy results showed that Cryptosporidium infections were associated with diarrhea, only infections with C. canis, C. felis, and subtype family Id of C. hominis were associated with diarrhea, and infection with C. parvum was associated with chronic diarrhea and vomiting. These results demonstrate that different Cryptosporidium genotypes and subtype families are linked to different clinical manifestations.

Cryptosporidium Species and Genotypes in HIV-Positive Patients in Lima, Peru

ABSTRACT: Cryptosporidium parasites from a cross‐sectional study conducted in two national hospitals in Lima, Peru were genetically characterized to deteimine the diversity of Cryptosporidium spp. in HIV‐positive people. A total of 2,672 patients participated in this study and provided 13,937 specimens. Cryptosporidium oocysts were detected by microscopy in 354 (13.3%) of the patients. Analysis of 951 Cryptosporidium‐ positive specimens from 300 patients using a small subunit rRNA‐based PCR‐RFLP tool identified 6 genotypes; Cryptosporidium hominis was the species most frequently detected (67.5%), followed by C. meleagridis (12.6%) and C. parvum (11.3%). Cryptosporidium canis (4.0%), C. felis (3.3%), and Cryptosporidium pig genotype (0.5%) were also found. These findings indicate that C. hominis is the predominant species in Peruvian HIV‐positive persons, and that zoonotic Cryptosporidium spp. account for about 30% of cryptosporidiosis in these patients.

Microscopic Observation Drug Susceptibility Assay, a Rapid, Reliable Diagnostic Test for Multidrug-Resistant Tuberculosis Suitable for Use in Resource-Poor Settings

ABSTRACT There is an urgent need for new tools to improve our ability to diagnose tuberculosis (TB) and multidrug-resistant TB (MDR-TB) in resource-poor settings. In a retrospective analysis undertaken in a region with a high incidence of TB, we evaluated the performance of the microscopic observation drug susceptibility assay (MODS), a novel assay developed in Perú which uses an inverted light microscope and culture in Middlebrook 7H9 broth to detect mycobacterial growth. MODS detected 94.0% of 1,908 positive sputum cultures, whereas Löwenstein-Jensen (LJ) culture detected only 86.9% (P < 0.001). The median time to culture positivity was 8 days (compared to 16 days for the same 208 samples by LJ culture; P < 0.001, Wilcoxon signed rank test). The results obtained by direct susceptibility testing using MODS demonstrated excellent concordance for isoniazid and rifampin and the detection of multidrug resistance with those obtained by indirect colorimetric methods: the microplate Alamar Blue assay (MABA) and the tetrazolium microplate assay (TEMA) (agreement, 95, 98, and 94%; kappa values, 0.8, 0.7, and 0.7, respectively). The concordance of the susceptibility testing results for ethambutol and streptomycin was poor. MODS is a novel assay which can detect the organisms responsible for TB and MDR-TB directly from sputum inexpensively, rapidly, and effectively. A comprehensive prospective evaluation of MODS is under way in Perú, and independent validation in nonresearch laboratories should be undertaken at the earliest opportunity.

The Epidemiology of Intestinal Microsporidiosis in Patients with HIV/AIDS in Lima, Peru

We studied microsporidiosis in human immunodeficiency virus-positive patients in 2 Lima hospitals. Of 2652 patients, 66% were male, 6% received antiretroviral therapy (ART), and the median CD4 lymphocyte count was 131 cells/microL. Sixty-seven patients (3%) had microsporidiosis; stool specimens from 56 were identified as having Enterocytozoon bieneusi of 10 different genotypes. The 2 most common genotypes, Peru-1 and Peru-2, were not associated with significant increases in chronic diarrhea; other genotypes were associated with a 4-fold increased risk. Risk factors for E. bieneusi infection segregated by genotype: contact with duck or chicken droppings and lack of running water, flush toilet, or garbage collection with genotype Peru-1 and watermelon consumption with other genotypes. Shortened survival was associated with low CD4 lymphocyte count (P<.0001), no ART (P<.0001), and cryptosporidiosis (P=.004) but not with microsporidiosis (P=.48). Our data suggest the possibility of zoonotic E. bieneusi transmission and an association with poor sanitary conditions.

A Molecular Biologic Study of Enterocytozoon bieneusi in HIV-Infected Patients in Lima, Peru

ABSTRACT. A cross‐sectional study was conducted to examine the genotype distribution of Enterocytozoon bieneusi in HIV‐infected patients who visited two government hospitals in Lima, Peru from January 2000 through March 2003. Microsporidia were detected by microscopy in 105 (3.9%) of 2,672 patients. A total of 212 stool samples from 89 microsporidia‐positive patients were genotyped by sequence analysis of the internal transcribed spacer (ITS) region of the rRNA gene. A 392‐bp fragment containing the complete ITS region was amplified and sequenced. Multiple alignments and phylogenetic analysis of these ITS sequences identified 11 distinct genotypes of E. bieneusi (Peru‐1 to Peru‐11), 6 of which were new genotypes not reported before. The remaining 5 genotypes had nucleotide sequences identical to those previously reported in humans, cats, pigs, and wild mammals. All the 11 E. bieneusi‐genotypes identified are genetically related, and members of the group have been previously found in humans, domestic animals, and some wild mammals. Thus, there is a high genetic diversity of E. bieneusi in humans in Peru, and zoonotie transmission is possible if humans are in close contact with infected animals.

Diagnosis of sputum-scarce HIV-associated pulmonary tuberculosis in Lima, Peru

Sputum induction, bronchoalveolar lavage, or gastric aspiration are often needed to produce adequate diagnostic respiratory samples from people with HIV in whom tuberculosis is suspected. Since these procedures are rarely appropriate in less-developed countries, we compared the performances of a simple string test and the gold-standard sputum induction. 160 HIV-positive adults under investigation for tuberculosis, and 52 asymptomatic HIV-positive control patients underwent the string test followed by sputum induction. The string test detected tuberculosis in 14 patients in whom this disease was suspected; sputum induction detected only eight of them (McNemars test, p=0.03). These preliminary data suggest that the string test is safe and effective for retrieval of useful clinical specimens for diagnosis of pulmonary tuberculosis, and is at least as sensitive as sputum induction.

Purification of Enterocytozoon bieneusi Spores from Stool Specimens by Gradient and Cell Sorting Techniques

ABSTRACT A three-step method for the purification of Enterocytozoon bieneusi spores from stool specimens was developed. The primary process of purification of the spores from bacterial contaminants involved Percoll gradient centrifugation followed by additional separation using cesium chloride density gradient centrifugation. The cesium chloride-isolated spores were further purified using a flow cytometer with cell sorting capabilities. Sorting was performed without the use of antibodies, fluorochromes, or dyes, leaving the sorted spores in their native state, which appears to be less destructive for spores. When quantified by flow cytometry using tubes with known numbers of highly fluorescent polystyrene beads, the sorted material showed a slight decrease in light scatter characteristics compared with the slightly larger Encephalitozoon species spores. Although the overall recovery of the E. bieneusi spores was low, calcofluor and Gram chromotrope staining, indirect immunofluorescence assay, and transmission electron microscopy revealed that the sorted material was highly purified and contained large numbers of E. bieneusi spores and relatively few bacteria and other debris. The sorted material appeared to be sufficiently pure and could be used for in vitro culture and for the development of a variety of diagnostic reagents as well as in studying the genome of E. bieneusi and host-parasite interactions.

Appropriate technology in tuberculosis diagnostics.

First, the statement that the use of the string test “will be hampered by the need for sputum induction” seems to be a misunderstanding: sputum induction added nothing to the results of the string test and was used only as a comparative test for this research. In fact, our findings clearly show that the string test alone offers better diagnostic sensitivity than sputum induction. By obviating the need for sputum induction, it could remove an important risk factor for nosocomial transmission of tuberculosis, particularly in resource-poor settings with a high tuberculosis burden, no isolation facilities, and wards crowded with highly susceptible HIV-infected patients. We would therefore suggest that the string test should supersede sputum induction if these data are borne out in other settings.