Arthur Clinton White

Nucleic Acid Test to Diagnose Cryptosporidiosis: Lab Assessment in Animal and Patient Specimens

Diarrheal diseases cause more morbidity and mortality around the world than human immunodeficiency virus (HIV), malaria, or tuberculosis. Given that effective treatment of persistent diarrheal illness requires knowledge of the causative organism, diagnostic tests are of paramount importance. The protozoan parasites of the genus Cryptosporidium are increasingly recognized to be responsible for a significant portion of diarrhea morbidity. We present a novel nucleic acid test to detect the presence of Cryptosporidium species in DNA extracted from stool samples. The assay uses the isothermal amplification technique recombinase polymerase amplification (RPA) to amplify trace amounts of pathogen DNA extracted from stool to detectable levels in 30 min; products are then detected visually on simple lateral flow strips. The RPA-based Cryptosporidium assay (RPAC assay) was developed and optimized using DNA from human stool samples spiked with pathogen. It was then tested using DNA extracted from the stool of infected mice where it correctly identified the presence or absence of 27 out of 28 stool samples. It was finally tested using DNA extracted from the stool of infected patients where it correctly identified the presence or absence of 21 out of 21 stool samples. The assay was integrated into a foldable, paper and plastic device that enables DNA amplification with only the use of pipets, pipet tips, and a heater. The performance of the integrated assay is comparable to or better than polymerase chain reaction (PCR), without requiring the use of thermal cycling equipment. This platform can easily be adapted to detect DNA from multiple pathogens.

Recombinase polymerase amplification-based assay to diagnose Giardia in stool samples.

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance.

Pre-assembled ssRNA-Argonaute complexes: a novel method to silence genes in Cryptosporidium

Cryptosporidiosis is a common cause of diarrhea morbidity and mortality worldwide. Research progress on this infection has been slowed by lack of methods to genetically manipulate Cryptosporidium parasites. Small interfering RNA (siRNA) is widely used to study gene function, but Cryptosporidium species lack the enzymes necessary to process siRNA. By preassembling complexes with the human enzyme Argonaute 2 (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA), we induced specific slicing in Cryptosporidium RNA targets. We demonstrated the reduction in expression of target genes at the mRNA and protein levels by transfecting live parasites with ssRNA-hAgo2 complexes. Furthermore we used this method to confirm the role of selected molecules during host cell invasion. This novel method provides a novel means of silencing Cryptosporidium genes to study their role in host-parasite interactions and as potential targets for chemotherapy.

Amplification-Free Detection of Cryptosporidium parvum Nucleic Acids with the Use of DNA/RNA-Directed Gold Nanoparticle Assemblies

Abstract:   This study describes the development and evaluation of an amplification-free molecular assay for detection of Cryptosporidium parvum oocysts. The assay employed a pair of oligonucleotide-functionalized gold nanoparticle (AuNP) probes that were complementary to adjacent sequences on C. parvum 18s rRNA. Hybridization of the probes to the target RNA resulted in the assembly of AuNPs into target-linked networks, which were detected both visibly and spectroscopically, by a redshift in the wavelength of light scattered by the gold nanoparticles. The limit of detection was between 4 × 105 and 4 × 106 copies of RNA per microliter reaction mix, when a short synthetic target or full-length in vitro transcribed target was employed. With total nucleic acids purified from C. parvum oocysts spiked into 100-mg stool, as few as 670 oocysts/μl reaction mix were detected. The ability to detect the nucleic acids of C. parvum oocysts in stool, without the need for complex amplification, offers unique advantages for such AuNP aggregation assays to be extended toward use in resource-limited settings where protozoan detection is needed most.

Molecular diagnosis of protozoan parasites by Recombinase Polymerase Amplification

Infections caused by protozoan parasites affect millions of people around the world. Traditionally, diagnosis was made by microscopy, which is insensitive and in some cases not specific. Molecular methods are highly sensitive and specific, but equipment costs and personnel training limit its availability only to specialized centers, usually far from populations with the highest risk of infection. Inexpensive methods that can be applied at the point of care (POC), especially in places with limited health infrastructure, would be a major advantage. Isothermal amplification of nucleic acids does not require thermocyclers and is relatively inexpensive and easy to implement. Among isothermal methods, recombinase polymerase amplification (RPA) is sensitive and potentially applicable at POC. We and others have developed RPA diagnostic tests to detect protozoan parasites of medical importance. Overall, our results have shown high specificity with limits of detection similar to PCR. Currently, the optimization of RPA for use at the POC is under development, and in the near future the tests should become available to detect protozoan infections in the field. In this review we discuss the current status, challenges, and future of RPA in the field of molecular diagnosis of protozoan parasites.

Fasciola hepatica Infection in an Indigenous Community of the Peruvian Jungle

Fasciola hepatica is a zoonotic infection with a worldwide distribution. Autochthonous cases have not been reported in the Amazon region of Peru. Operculated eggs resembling F. hepatica were identified in the stools of five out of 215 subjects in a remote indigenous community of the Peruvian jungle. Polymerase chain reaction targeting Fasciola hepatica cytochrome oxidase subunit 1 (COI) gene and sequencing of the products confirmed Fasciola infection.

Advice on Malaria and Yellow Fever Prevention Provided at Travel Agencies in Cuzco, Peru

BACKGROUND Travelers receive medical advice from a variety of sources, including travel agencies. The aim of this study is to describe the quality of pre-travel advice provided by travel agencies in Cuzco to travelers interested in visiting malaria and yellow fever endemic areas. METHODS Trained medical students posed as tourists and visited travel agencies in Cuzco requesting travel advice for a trip to the southern Amazon of Peru, recording advice regarding risk and prevention of malaria and yellow fever. RESULTS A total of 163 registered travel agencies were included in the study. The mean proposed tour duration was 6.8 days (±1.4 days) with a median time to departure of 3 days and a median tour cost of 805 US dollars (USD) [interquartile range (IQR) 580-1,095]. Overall, 45% employees failed to mention the risk for any illness. Eighteen percent of the employees acknowledged risk of malaria and 53% risk of yellow fever. However, 36% denied malaria risk and 2% denied risk of yellow fever in the region. The price of tours from travel agencies that did not mention any health risk was significantly lower [1,009.6 ± 500.5 vs 783.9 ± 402 USD, t (152) = 3, p < 0.01] compared with the price from agencies that did mention health risks. Almost all who acknowledged malaria (97%) and/or yellow fever (100%) were able to provide at least one recommendation for prevention. However, advice was not always accurate or spontaneously volunteered. Only 7% of the employees provided both correct scheduling and location information for administration of the yellow fever vaccine. CONCLUSIONS The majority of registered travel agencies in Cuzco did not provide sufficient and accurate information regarding risk and prevention of malaria and yellow fever to travelers inquiring about trips to the southern Amazon of Peru.

CADM1 Overexpression and CD7 Downregulation in Strongyloides stercoralis and HTLV-1 Coinfection as Possible Markers for Adult T-cell Leukemia/Lymphoma progression.

Abstract Background Human T-cell lymphotropic virus (HTLV-1) is a retrovirus endemic in Latin America, Africa, and Asia. Increasing numbers are being reported in the United States. HTLV-1 causes Adult T-cell Leukemia/Lymphoma (ATL) in 3–5% of HTLV-1 carriers, usually only after a prolonged latent period. Co-infection with the nematode Strongyloides stercoralis (SS) is associated with early onset of ATL. The exact mechanism by which SS accelerates ATL development in HTLV-1 subjects is not understood. CADM1 has been recently identified as a surface marker of HTLV-1 infected cells; CD7 is a probable marker of early cell transformation. We hypothesize that previous SS infection will increase the proportion of infected T cells (CADM1+) and lead to transformation of infected cells (CADM1+CD7low) in HTLV-1 subjects. Methods In this pilot study, we tested seven subjects that were diagnosed with HTLV-1 between 2006 and 2008 and actively followed up at the HTLV-1 Cohort Clinic at the Tropical Medicine Institute in Lima, Peru. Five had previous SS infection. We performed surface flow cytometry staining of peripheral blood mononuclear cells with monoclonal antibodies against CADM1, CD7, CD4, and CD3. Current SS infection was ruled out by the Baermann technique in a stool sample. Results Average age was 53 years (range 30–79). Average time since diagnosis of HTLV-1 was 10 years (range 9–11). One 30-year-old patient with prior SS infection was diagnosed with ATL and hospitalized in the ICU at the time of follow-up and excluded from group comparison. There was a trend for higher proportions of CADM1+ T-cells in subjects with previous SS co-infection compared with those without SS (median 16.20% vs. 9.55%, P = 0.13, Mann–Whitney). The proportion of PBMCs that were CADM1+CD7− was also increased in some of those with previous SS (Figure 1). Conclusion Our pilot data suggest that SS co-infection is associated with prolonged effects on HTLV-1 infection. We noted trends towards increased numbers of HTLV-1 infected cells (CADM1+) and a predisposition towards malignant transformation (CADM1+CD7low). Further studies are needed to confirm this observation and to determine whether CADM1 and CD7 expression may be useful as a screening tool to monitor HTLV-1 subjects at high risk of developing ATL.Figure 1. Representative flow cytometry. Disclosures All authors: No reported disclosures.

Kato-Katz and Lumbreras rapid sedimentation test to evaluate helminth prevalence in the setting of a school-based deworming program

The sensitivity of the Kato-Katz test is suboptimal for the evaluation of intestinal helminth prevalence. Moreover, during mass deworming, as helminth egg burden decreases, the sensitivity is likely to decrease. The Lumbreras rapid sedimentation (Lumbreras) is a low-cost non-quantitative test, but may provide useful information in low burden areas. We compared the prevalence of intestinal helminth infections assessed by the Kato-Katz and the Lumbreras rapid sedimentation test on 3 stool specimens from each of 1083 children. The sensitivities were compared using the McNemar paired test. Using the combined outcome of the 3 different stool tests as the standard, Kato-Katz had lower sensitivity than Lumbreras rapid sedimentation tests for Ascaris lumbricoides (85.1% vs. 95.1%, p = 0.03), Hymenolepis nana (77.7% vs. 97.9%, p < 0.01), Trichuris trichura (41.7% vs. 100%, p = 0.01), hookworm (0% vs. 100%, p = 0.01), and Strongyloides stercoralis (0% vs. 88%, p < 0.01). Kato-Katz demonstrated significantly lower sensitivity, missing most T. trichiura, hookworm, and S. stercoralis infections. The combination of Kato-Katz and Lumbreras rapid sedimentation tests enables the detection of more intestinal helminths infections in post-deworming low prevalence areas.

Determining better treatments for neurocysticercosis

Neurocysticercosis, caused by Taenia solium, is emerging as a major cause of neurological disease worldwide. Despite the availability of antiparasitic drugs for this infection for more than 35 years, the role of antiparasitic drugs in management remains controversial. In these 35 years, several controlled and uncontrolled trials have attempted to clarify the role of antiparasitic drugs in the management of neurocysticercosis; data from these trials were mostly confl icting. Most controversies were due to poorly collected and uncontrolled data. Strangely, the controversies revolved around to treat or not to rather than how to optimally treat? In 2004, Garcia and colleagues reported the results of a randomised placebo-controlled trial of albendazole (800 mg per day for 7 days) in viable parenchymal cysticercosis. At 6 months, nearly 35% of participants given albendazole were free of viable cysts compared with 13% of those given placebo. The results of the trial clearly showed the benefi ts of albendazole treatment in terms of cyst resolution. Even so, viable cysts persisted in nearly 65% of those given antiparasitic treatment with albendazole. When the total number of viable cysts persisting at 6 months were analysed, 41% of the cysts remained viable despite albendazole treatment, prompting the search for better treatment regimens with the available antiparasitic drugs. Several options were considered to improve parasiticidal activity. lncreased duration of treatment with albendazole was considered, but was shown to not off er any advantage over the standard 7-day course, specifi cally in viable parenchymal cysticercosis. Other options were a repeat course of albendazole and use of increased doses of albendazole, which was reported to be associated with increased parasiticidal activity in one study. Finally, another option considered was combination anthelmintic treatment with albendazole and praziquantel given simultaneously. The theoretical premise for combination treatment was provided by the diff erent mechanisms of action of the two drugs. The combination had been tried previously in cystic echinococcosis and also parenchymal cysticercosis in small trials. ln this issue of The Lancet lnfectious Diseases, Garcia and colleagues report the results of a rigorously conducted randomised, active-comparator trial of combination antiparasitic treatment. The three arms of the trial were standard albendazole dose (maximum 800 mg per day), increased albendazole dose (maximum 1200 mg per day), and combination albendazole and praziquantel (in standard doses). Combination antiparasitic treatment was associated with a signifi cantly increased proportion of participants with complete resolution (64% compared with 37% in standard albendazole group), showing a signifi cantly improved rate of parasite clearance. A previous study by the investigators assessed the pharmacokinetics of the combination regimen and reported increased serum albendazole concentrations raising the question of whether the improved resolution associated with combination treatment was due to the eff ects of combination or only increased albendazole effi cacy. The report combined two diff erent study designs addressing two somewhat disparate issues: (1) a randomised controlled trial comparing the three diff erent regimens, and (2) a cohort design, which addressed the association between cyst resolution and seizure incidence. ln the randomised study, combination therapy was associated with more rapid resolution of lesions. ln the cohort study, resolution of infestation was signifi cantly associated with reduced seizure recurrence even after adjustment for treatment regimens. These results resolve some questions but raise others. For example, does the choice of treatment (combined or enhanced albendazole treatment versus Lancet Infect Dis 2014