Anton M. Polderman

Simultaneous Detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in Fecal Samples by Using Multiplex Real-Time PCR

ABSTRACT 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.

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.

Immunodiagnosis of schistosomiasis by determination of the circulating antigens CAA and CCA, in particular in individuals with recent or light infections

In the present paper, we evaluate determination of circulating anodic (CAA) and cathodic (CCA) antigen for the diagnosis of an active Schistosoma infection in humans, in comparison to the diagnostic performance of parasitological examination and the demonstration of specific antibodies. Illustrated by three different studies, which all deal with the diagnosis of either recent or low intensity infections, we further discuss our experiences with these diagnostic methods. For the diagnosis of recent infections, specific antibody determination showed to be very sensitive, particularly in individuals originating from non-endemic areas. For the assessment of cure and for the diagnosis of active infections in endemic areas, the methods of choice are parasitological examination and CAA or CCA determination. Depending on infection levels of the target population and on logistic conditions, CAA and CCA determination may either replace parasitological examination or, in the case of light infections, may be used as a complementary diagnostic tool.

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 circulating anodic antigen by ELISA for seroepidemiology of schistosomiasis mansoni

Sera of individuals from Burundi excreting eggs of Schistosoma mansoni (prevalence 35%; 178 subjects) and of similar individuals from Maniema, Zaire (prevalence 95%; 99 subjects), and of 159 Dutch and 81 Zairean non-infected controls, were screened by enzyme-linked immunosorbent assay for the presence of schistosome circulating anodic antigen (CAA). No false positive results were obtained. The sensitivity of the test was 75% in Burundi and 93% in Zaire, a significant difference (P less than 0.05). However, in matched egg output classes the test results did not differ significantly; 60% and 67%, respectively, of those excreting 1-100 eggs per gram of faeces (epg), 86% and 100% of those excreting 101-400 epg, and 100% of those excreting over 400 epg were detected. The efficiency of the assay was 91% in Burundi and 93% in Zaire. The Spearman rank coefficient of correlation between antigen titre and egg output (determined by 3 consecutive Kato egg counts) was 0.61 in Burundi and 0.82 in Zaire. The sensitivity of the test compared well with a single egg count. In addition, preliminary data showed that occasionally CAA was detectable in serum of individuals not excreting schistosome eggs. As CAA is found only in the presence of living worms, such cases reflect active infections.

Determining the prevalence of Oesophagostomum bifurcum and Necator americanus infections using specific PCR amplification of DNA from faecal samples

Until recently infection of humans with Oesophagostomum bifurcum was regarded as a rare zoonosis. But in northern Togo and Ghana its prevalence is 50% or more in certain villages. Diagnosis is hampered by the fact that the eggs of O. bifurcum are morphologically identical to those of the hookworm Necator americanus. Stools have to be cultured for 7 days to allow eggs to hatch to the characteristic third‐stage (L3) larvae. We evaluated the applicability of specific polymerase chain reactions (PCRs) to amplify DNA from faecal samples as an alternative method for the differential diagnosis of the two infections. Oesophagostomum bifurcum‐PCR was positive in 57 of 61 faecal samples known to contain O. bifurcum L3 larvae in coproculture. Necator americanus PCR was positive in 137 of 146 faecal samples known to contain N. americanus L3 larvae in coproculture. PCR also detected 26 additional O. bifurcum cases in 72 samples from O. bifurcum endemic villages in which no O. bifurcum larvae were found and 45 N. americanus cases in 78 samples in which no N. americanus larvae were found in coproculture. No O. bifurcum DNA was detected in 91 stool samples from individuals from two non‐endemic villages. These results prove the usefulness of specific PCR assays as epidemiological tools to estimate the prevalence of O. bifurcum and N. americanus infections in human populations.

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%).

A model for variations in single and repeated egg counts in Schistosoma mansoni infections

Faecal egg counts are often used to measure Schistosoma mansoni infection, but the considerable variation between successive counts complicates their interpretation. The stochastic model described in this paper gives a description of observed egg counts in a population and can be used as a tool to gain an insight into the underlying worm load distribution. The model distinguishes between two sources of variation in egg counts: (1) variation caused by the difference in worm load between individuals, and (2) the variability of egg counts for an individual with a given worm load. Empirical data, single and repeated measurements, from surveys in five villages in Burundi and Zaire have been used to fit and validate the model. We have discussed possible mechanisms that explain the differences in estimated values between the villages. The model indicates that the expected number of eggs in a stool sample per S. mansoni worm pair is lower than suggested by autopsy data and that, possibly as a consequence of immunity, the inter-individual variation in worm loads decreases with age.

Genetic Variation among Human Isolates of Uninucleated Cyst-Producing Entamoeba Species

ABSTRACT Twelve human infections with Entamoeba spp. producing uninucleated cysts were studied. DNA was extracted from infected feces and used to amplify part of the ameba small-subunit rRNA gene. Sequence analysis identified four distinct types ofEntamoeba, all of which are related toEntamoeba polecki and E. chattoni and two of which have not been reported previously. Whether these genetic types represent different species is unclear. We propose that the agent of all human infections with uninucleated cyst-producingEntamoeba species be reported as “E. polecki-like.”

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 womens 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.