Simultaneous detection and quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples using multiplex real-time PCR.

A multiplex real-time PCR was developed and evaluated for the simultaneous detection of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples. Using well-defined control samples (N = 150), known positive fecal samples (N = 50), and fecal samples from an area in Ghana where human infections with all 3 nematode species are endemic (N = 339), the method proved to be highly specific and sensitive. Cycle threshold (Ct) values, reflecting parasite-specific DNA load, showed significant correlation with the intensity of infection as measured by microscopy using Kato-Katz fecal smears or by species specific third-stage larval count after coproculture. The multiplex real-time PCR described combined with the simple fecal sample collection procedure and the potential for high throughput makes this approach a powerful diagnostic tool to study species-specific transmission patterns of human hookworm-like infections. Moreover, this procedure facilitates monitoring of intervention programs and allows species-specific detection of treatment failure following rounds of mass treatment.

Diagnosing Polyparasitism in a High-Prevalence Setting in Beira, Mozambique: Detection of Intestinal Parasites in Fecal Samples by Microscopy and Real-Time PCR.

BACKGROUND: Many different intestinal parasite species can co-occur in the same population. However, classic diagnostic tools can only frame a particular group of intestinal parasite species. Hence, one or two tests do not suffice to provide a complete picture of infecting parasite species in a given population. The present study investigated intestinal parasitic infections in Beira, Mozambique, i.e. in the informal settlement of Inhamudima. Diagnostic accuracy of five classical microscopy techniques and real-time PCR for the detection of a broad spectrum of parasites was compared. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional population-based survey was performed. One stool sample per participant (n = 303) was examined by direct smear, formal-ether concentration (FEC), Kato smear, Baermann method, coproculture and real-time PCR. We found that virtually all people (96%) harbored at least one helminth, and that almost half (49%) harbored three helminths or more. Remarkably, Strongyloides stercoralis infections were widespread with a prevalence of 48%, and Ancylostoma spp. prevalence was higher than that of Necator americanus (25% versus 15%), the hookworm species that is often assumed to prevail in East-Africa. Among the microscopic techniques, FEC was able to detect the broadest spectrum of parasite species. However, FEC also missed a considerable number of infections, notably S. stercoralis, Schistosoma mansoni and G. intestinalis. PCR outperformed microscopy in terms of sensitivity and range of parasite species detected. CONCLUSIONS/SIGNIFICANCE: We showed intestinal parasites-especially helminths-to be omnipresent in Inhamudima, Beira. However, it is a challenge to achieve high diagnostic sensitivity for all species. Classical techniques such as FEC are useful for the detection of some intestinal helminth species, but they lack sensitivity for other parasite species. PCR can detect intestinal parasites more accurately but is generally not feasible in resource-poor settings, at least not in peripheral labs. Hence, there is a need for a more field-friendly, sensitive approach for on-the-spot diagnosis of parasitic infections.

Intestinal microsporidiosis in diarrheal patients infected with human immunodeficiency virus-1 in Addis Ababa, Ethiopia.

Intestinal microsporidiosis is most commonly associated with persistent diarrhea in advanced AIDS cases. To determine the prevalence and clinical manifestations of this infection in HIV/AIDS patients, a single fresh stool sample and blood were collected from 243 (214 HIV-positive and 29 HIV-negative) diarrheal patients. The presence of intestinal microsporidiosis in the stool was determined by Uvitex-2B staining and a PCR-based detection method. HIV screening was done by using ELISA, and reactive samples were confirmed by Western blotting. The CD4+ cell count was analyzed using FACScan. Out of 243 diarrheal patients, 39 (16.0%) cases were positive for intestinal microsporidial infection by either of the methods used. Of the 39, only 18 cases positive by microscopy were also positive by PCR. Based on PCR and microscopic analyses the microsporidial parasites were identified as Enterocytozoon bieneusi (30), Ecephalitozoon intestinalis (6), and double infections (3). All microsporidia-positive cases were HIV-positive, and 92.3% had diarrhea for over 4 weeks. The diarrhea was watery in 79.5% of the patients. Weight loss >10% was recorded in 37 (94.9%) cases. The CD4+ cell count was <100 cells/mm(3) in 84.4% of subjects, and 59.4% of the patients had a CD4+ cell count of < or =50 cells/mm(3), with a mean of 22.8 cells/mm(3). This study revealed that intestinal microsporidiosis is a common cause of chronic diarrhea and severe weight loss in advanced AIDS patients in Ethiopia. This condition is attributable mainly to E. bieneusi. Thus, early diagnosis of intestinal microsporidiosis in HIV/AIDS patients would certainly be helpful in the understanding and management of diarrheal illness.

Detection of intestinal microsporidiosis in diarrhoeal patients infected with the human immunideficiency virus (HIV-1) using PCR and Uvitex-2B stain.

A total of 105 single fresh stool samples were collected from diarrhoeal patients with (80 HIV-positive and 25 HIV-negative) from the Army and the Police hospitals, Addis Ababa. The stool samples were processed by water-ether sedimentation method; they were stained with Uvitex-2B technique for microscopic detection of intestinal microsporidium. A portion of all samples were preserved in 200microl PBS containing 2% PVPP ((Polyvinylpolypyrolidone) for confirmation with PCR. 18/105(17.2%) of the cases were positive for intestinal microsporidial infection by at least one method. 8/105 (7.6%) positive both by microscopy and PCR and 10/105 (9.5%) were positive only by PCR. All microsporidia positive cases were also HIV positive. Based on PCR analysis, 15 Enterocytozoon bieneusi and 3 Encephalitozoon intestinalis were identified. This study has shown that intestinal microsporidiosis is a common cause of chronic diarrhoea in advanced AIDS patients and this is mainly attributed to Enterocytozoon bieneusi. To the best of our knowledge, this is the first report of intestinal microsporidiosis in Ethiopia. It has an important implication for the understanding of the aetiology of diarrhoea in HIV/AIDS patients in the country.

Spatial and temporal distribution of soil-transmitted helminth infection in sub-Saharan Africa: a systematic review and geostatistical meta-analysis.

BACKGROUND: Interest is growing in predictive risk mapping for neglected tropical diseases (NTDs), particularly to scale up preventive chemotherapy, surveillance, and elimination efforts. Soil-transmitted helminths (hookworm, Ascaris lumbricoides, and Trichuris trichiura) are the most widespread NTDs, but broad geographical analyses are scarce. We aimed to predict the spatial and temporal distribution of soil-transmitted helminth infections, including the number of infected people and treatment needs, across sub-Saharan Africa. METHODS: We systematically searched PubMed, Web of Knowledge, and African Journal Online from inception to Dec 31, 2013, without language restrictions, to identify georeferenced surveys. We extracted data from household surveys on sources of drinking water, sanitation, and women's level of education. Bayesian geostatistical models were used to align the data in space and estimate risk of with hookworm, A lumbricoides, and T trichiura over a grid of roughly 1 million pixels at a spatial resolution of 5 × 5 km. We calculated anthelmintic treatment needs on the basis of WHO guidelines (treatment of all school-aged children once per year where prevalence in this population is 20-50% or twice per year if prevalence is greater than 50%). FINDINGS: We identified 459 relevant survey reports that referenced 6040 unique locations. We estimate that the prevalence of hookworm, A lumbricoides, and T trichiura among school-aged children from 2000 onwards was 16·5%, 6·6%, and 4·4%. These estimates are between 52% and 74% lower than those in surveys done before 2000, and have become similar to values for the entire communities. We estimated that 126 million doses of anthelmintic treatments are required per year. INTERPRETATION: Patterns of soil-transmitted helminth infection in sub-Saharan Africa have changed and the prevalence of infection has declined substantially in this millennium, probably due to socioeconomic development and large-scale deworming programmes. The global control strategy should be reassessed, with emphasis given also to adults to progress towards local elimination. FUNDING: Swiss National Science Foundation and European Research Council.

Genetic substructuring within Oesophagostomum bifurcum (Nematoda) from human and non-human primates from Ghana based on random amplified polymorphic DNA analysis.

Random amplified polymorphic DNA (RAPD) was used to study genetic variation within Oesophagostomum bifurcum in Ghana. Four different decamer primers were used for the amplification of DNA from individual O. bifurcum adults (n = 41) from humans and non-human primates (including the Mona monkey, Patas monkey and Olive baboon) from different geographic regions. Analysis of the amplicons from all 41 nematodes by high resolution, denaturing polyacrylamide gel electrophoresis defined a total of 326 informative RAPD bands. Cluster analysis of the RAPD data (based on pairwise comparison of banding profiles) showed that O. bifurcum from humans was genetically distinct from O. bifurcum from the Mona and Patas monkeys, and from the Olive baboon. These findings clearly demonstrate the existence of population genetic substructuring within O. bifurcum from different primate hosts in Ghana, and raise interesting questions about host specificity, epidemiology (e.g., zoonotic transmission), and ecology of the different genotypes of O. bifurcum.

Molecular diagnosis of Strongyloides stercoralis in faecal samples using real-time PCR.

A real-time PCR method targeting the small subunit of the rRNA gene was developed for the detection of Strongyloides stercoralis DNA in faecal samples, including an internal control to detect inhibition of the amplification process. The assay was performed on a range of well-defined control samples (n=145), known positive faecal samples (n=38) and faecal samples from a region in northern Ghana where S. stercoralis infections are highly endemic (n=212), and achieved 100% specificity and high sensitivity. The use of this assay could facilitate monitoring the prevalence and intensity of S. stercoralis infections during helminth intervention programs. Moreover, the use of this assay in diagnostic laboratories could make the introduction of molecular diagnostics feasible in the routine diagnosis of S. stercoralis infections, with a two-fold increase in the detection rate as compared with the commonly used Baermann sedimentation method.

Impact of repeated mass treatment on human Oesophagostomum and hookworm infections in northern Ghana.

Oesophagostomum bifurcum is a common parasite of humans causing disease in parts of northern Ghana and northern Togo. The impact of repeated mass treatment with albendazole on infection with O. bifurcum and hookworm is analysed and the results compared with those in a control area where no treatment was given. At baseline, O. bifurcum and hookworm prevalences were 53.0% and 86.9%, respectively (n=1011). After 12 months, following two rounds of albendazole treatment, prevalences decreased significantly to 5.4% for O. bifurcum and 36.8% for hookworm (n=535). Twenty-four months after the baseline survey and following a total of four rounds of treatment, prevalences were further reduced to 0.8% and 23.4% for O. bifurcum and hookworm, respectively (n=478). Overall, there was a significant decrease in the larval counts, measured as geometric mean larval count per 4 g of stool of O. bifurcum from 3.0 to 0.1 and of hookworm from 47.2 to 1.8. The fourth mass treatment was carried out in April 2003 by the Lymphatic Filariasis Elimination Programme. Overall, compliance to treatment varied from 70% to 80%. In the control area, Oesophagostomum prevalence increased from 18.5% to 37.0% and the intensity from 0.4 to 1.4. For hookworm, both prevalence (86.1-91.3%) and intensity (54.8-74.3) increased but not to a significant level. The prospects of eliminating human oesophagostomiasis from the intervention area, while simultaneously achieving an important reduction of hookworm prevalences by albendazole mass treatment, are discussed.

AFLP fingerprinting for the analysis of genetic diversity within Necator americanus.

In the present study, we utilised the method of AFLP to screen for genetic variation within and among individuals of the blood-feeding human hookworm Necator americanus (Nematoda) from Africa, Asia and South America. A total of 45 adult worms (i.e. 20 from Ghana, 16 from Colombia and 9 from Nepal) were subjected to analysis using the restriction enzyme/primer combination HindIII+AG/BglII+AC. Cluster analysis divided N. americanus into multiple, genetically distinct groups, consistent with previous findings using ribosomal and mitochondrial DNA data sets. The results demonstrated the usefulness of AFLP fingerprinting for establishing genetic variation within N. americanus and reinforce its applicability to other parasitic helminths of human and/or veterinary health importance.

Diagnostic methods for differentiation of Entamoeba histolytica and Entamoeba dispar in carriers: performance and clinical implications in a non-endemic setting.

Unpreserved faecal samples, suspected to contain Entamoeba histolytica/Entamoeba dispar cysts or trophozoites on the basis of microscopic examination, and serum samples from 416 patients were collected in a prospective study to determine whether stool antigen assays and detection of antibodies in serum are reliable methods to distinguish between carriers of E. histolytica and E. dispar in comparison to the reference test: real-time PCR. In 283 patients (68%) DNA of E. histolytica or E. dispar was amplified by real-time PCR: 6 patients with amoebic colitis (2%), 19 carriers of E. histolytica (6.7%), and 258 carriers of E. dispar (91.2%). In 133 patients (31%) no DNA of E. histolytica or E. dispar could be amplified in the stool samples. This patient group was used as control for the evaluation of diagnostic tests. Using real-time PCR as a reference test, the sensitivity and specificity of (1) the Entamoeba test for the diagnosis of E. histolytica/E. dispar carrier were 59% and 98%, (2) E. histolytica II for the diagnosis of E. histolytica carrier was 71% and 100%, and (3) serology for the diagnosis of E. histolytica infection was 83.3% and 95.2%, respectively. Applied to carriers that did not originate from an endemic country the sensitivity of serology for E. histolytica infection was 90% and specificity was 98.8%. In comparison to real-time PCR the performances of Entamoeba test and E. histolytica II lacked sensitivity for a reliable diagnosis of E. histolytica/E. dispar infection in a non-endemic setting. In carriers of E. histolytica/E. dispar from non-endemic countries the high specificity of serology can be used to establish the diagnosis of E. histolytica infection if antibodies are present.

Polymerase chain reaction-based differential diagnosis of Ancylostoma duodenale and Necator americanus infections in humans in northern Ghana.

We evaluated a two-step semi-nested polymerase chain reaction (PCR)-based approach for the specific detection of Ancylostoma duodenale DNA in human faeces. The test was used to determine to what extent this species of hookworm is present in the regions of Bolgatanga and Garu of northern Ghana. Initially, the sensitivity and specificity of the PCR were tested using a range of well-defined control samples. Subsequently, a total of 378 human faecal DNA samples from Bolgatanga and Garu were subjected to the PCR. The results were compared with those obtained using a previously established PCR for the specific detection of Necator americanus DNA in human faeces. Infection with A. duodenale was recorded in 74 (19.6%) samples and N. americanus in 278 (73.5%), of which 64 (16.9%), represented co-infections with both species. While A. duodenale was predominantly detected in the samples from Bolgatanga, infections in Garu related almost exclusively to N. americanus. The results showed that the present PCR approach is a valuable complementary tool for the diagnosis of A. duodenale infection in humans in Ghana, having implications for epidemiological studies and for the monitoring of the success of control programmes in regions in Africa.

Short communication: Misleading microscopy in amoebiasis.

High prevalences of intestinal amoebiasis are commonly reported by microscopy in Ethiopia. In order to confirm the actual occurrence of Entamoeba histolytica we collected 108 stool specimens from different hospitals & health centers from patients in whom haematophagous trophozoites were believed to be found. We detected only a single E. histolytica case while 77 (71.3%) were E. dispar and the remaining 30 samples were negative for both species by real-time PCR based on the small subunit ribosomal RNA gene sequence of E. histolytica and E. dispar. The tradition of microscopy in a routine diagnostic set-up appears unsatisfactory to reliably differentiate rbc-engulfing amoeba from non-invasive amoeba in wet smears.

Real-time PCR for the detection of Giardia lamblia.

Microscopy is considered to be the gold standard for diagnosis of Giardia lamblia infection. However, this method is time-consuming and not very sensitive. We developed a real-time PCR assay based on the small subunit ribosomal RNA gene of G. lamblia for the specific detection of G. lamblia DNA in stool samples and thereafter compared the results with microscopy and antigen detection. The G. lamblia real-time PCR was positive in 102 of 104 fecal samples known to contain G. lamblia cysts and was positive in 10 fecal samples in which G. lamblia antigen was detected but in which no cysts were found with microscopic examination of concentrated fecal samples. The real-time PCR is as specific and sensitive as antigen detection and is more sensitive than microscopy. Moreover, in two patients we were able to detect G. lamblia earlier in the course of infection than with any of the other methods.

Detection of Cyclospora cayetanensis in travellers returning from the tropics and subtropics using microscopy and real-time PCR.

We examined 100 stool specimens of returning travellers with diarrhoea for the presence of Cyclospora cayetanensis using fluorescence microscopy and real-time PCR. C. cayetanensis was found in four cases with microscopy and PCR. One additional sample was positive only by PCR, and could be confirmed by microscopic examination of several additional slides. C. cayetanensis was the most frequent parasitic cause of diarrhoea after Giardia duodenalis.

Detection and identification of entamoeba species in stool samples by a reverse line hybridization assay.

Classically, detection of Entamoeba histolytica is performed by microscopic examination for characteristic cysts and/or trophozoites in fecal preparations. Differentiation of E. histolytica cysts and those of nonpathogenic amoebic species is made on the basis of the appearance and the size of the cysts. However, by classical means objective tools for confirmation and quality control do not exist. Therefore, a reverse line blot hybridization assay was developed to detect a variety of Entamoeba species and genetic variants known to infect humans. The assay was performed after amplification with general Entamoeba-specific primers. The assay could identify four genetic variants of Entamoeba polecki-like cysts as well as E. histolytica, Entamoeba dispar, Entamoeba hartmanni, Entamoeba moshkovskii and Entamoeba coli and even mixed infections in a range of controls and fecal samples. This technique can be used as an additional standard for diagnosis, epidemiology, and quality control for amoebic infections.

Short communication: Prevalence of Entamoeba histolytica and Entamoeba dispar in northern Ghana.

Since the redescription of the potentially invasive Entamoeba histolytica, separating it from the morphologically identical non-invasive Entamoeba dispar, there is a need for the reassessment of epidemiological data on amoebiasis. In this context we conducted a descriptive survey on the presence of E. histolytica and E. dispar in a rural area in northern Ghana. We found a high prevalence (39.8%) of the E. histolytica/E. dispar complex with microscopy, but E. histolytica and E. dispar-specific DNA amplification using real-time polymerase chain reaction identified only one E. histolytica case and revealed a considerably higher prevalence of E. dispar (82.8%).

Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR.

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium are three of the most important diarrhea-causing parasitic protozoa. For many years, microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of E. histolytica, G. lamblia, and C. parvum infections. Recently, more specific and sensitive alternative methods (PCR, enzyme-linked immunosorbent assay, and direct fluorescent-antibody assay) have been introduced for all three of these parasitic infections. However, the incorporation in a routine diagnostic laboratory of these parasite-specific methods for diagnosis of each of the respective infections is time-consuming and increases the costs of a stool examination. Therefore, a multiplex real-time PCR assay was developed for the simultaneous detection of E. histolytica, G. lamblia, and C. parvum in stool samples. The multiplex PCR also included an internal control to determine efficiency of the PCR and detect inhibition in the sample. The assay was performed on species-specific DNA controls and a range of well-defined stool samples, and it achieved 100 percent specificity and sensitivity. The use of this assay in a diagnostic laboratory would provide sensitive and specific diagnosis of the main parasitic diarrheal infections and could improve patient management and infection control.

Entamoeba histolytica infections in captive primates.

A group based survey on the presence of Entamoeba histolytica and Entamoeba dispar using real-time PCR among 20 species of captive non-human primates was performed after diagnosis of E. histolytica dysentery in a spider monkey ( Ateles belzebuth hybridus). E. histolytica DNA was detected in three species of New World primates and in three species of Old World primates. In five of six E. histolytica isolates, it was possible to amplify the SREHP gene. They all revealed the same pattern after AluI digestion, indicating a common source of infection. E. dispar DNA was detected in two species of New World monkeys and three species of Old World monkeys. The results demonstrate that E. histolytica is capable of causing symptomatic and non-symptomatic infections in Old World and New World non-human primates. To our knowledge, this is the first report of E. histolytica sensu stricto in non-human primates after the redescription separating it from E. dispar in 1993.