Jaco J. Verweij

Molecular diagnostics of intestinal parasites in returning travellers.

A new diagnostic strategy was assessed for the routine diagnosis of intestinal parasites in returning travellers and immigrants. Over a period of 13xa0months, unpreserved stool samples, patient characteristics and clinical data were collected from those attending a travel clinic. Stool samples were analysed on a daily basis by microscopic examination and antigen detection (i.e. care as usual), and compared with a weekly performed multiplex real-time polymerase chain reaction (PCR) analysis on Entamoeba histolytica, Giardia lamblia, Cryptosporidium and Strongyloides stercoralis. Microscopy and antigen assays of 2,591 stool samples showed E. histolytica, G. lamblia, Cryptosporidium and S. stercoralis in 0.3, 4.7, 0.5 and 0.1% of the cases, respectively. These detection rates were increased using real-time PCR to 0.5, 6.0, 1.3 and 0.8%, respectively. The prevalence of ten additional pathogenic parasite species identified with microscopy was, at most, 0.5%. A pre-selective decision tree based on travel history or gastro-intestinal complaints could not be made. With increased detection rates at a lower workload and the potential to extend with additional parasite targets combined with fully automated DNA isolation, molecular high-throughput screening could eventually replace microscopy to a large extent.

Application of a circulating-cathodic-antigen (CCA) strip test and real-time PCR, in comparison with microscopy, for the detection of Schistosoma haematobium in urine samples from Ghana

Abstract In the detection of parasitic infection, the traditional methods based on microscopy often have low sensitivity and/or specificity compared with the newer, molecular tests. An assay based on real-time PCR and a reagent strip test for detecting circulating cathodic antigen (CCA) have both now been compared with urine filtration and microscopy, in the detection of Schistosoma haematobium infections. Urine samples, obtained from 74 cases in areas of Ghana with endemic S. haematobium and 79 controls from non-endemic areas, were each checked using the three methods. With the results of the filtration and microscopy taken as the gold standard, real-time PCR was found to be 100% specific and 89% sensitive whereas the CCA strips were 91% specific and 41% sensitive. With the samples found to contain ≥50 eggs/10 ml (indicating relatively intense infections), the sensitivities of the PCR and CCA were higher, at 100% and 62%, respectively. As expected, egg counts were negatively correlated with the number of amplification cycles needed, in the PCR, to give a signal that exceeded the background (r=−0.38; P<0.01). Although the real-time PCR and CCA strip tests are very different, both show promise in the detection of S. haematobium infections. The PCR has optimal specificity and high sensitivity but the specificity of the CCA strips and the sensitivity of both tools could still be improved. A more thorough re-evaluation of the sensitivity and specificity of microscopy and these newer diagnostic methods, with an estimation of the cost-effectiveness of each technique, is recommended.

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 7u2003days 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%).

Differentiation of Entamoeba histolytica and Entamoeba dispar cysts using polymerase chain reaction on DNA isolated from faeces with spin columns

Abstractu2002Since Entamoeba histolytica and Entamoeba dispar were formally recognized as two different species at the World Health Organization (WHO)/Pan American Health Organization (PAHO)/United Nations Educational, Scientific and Cultural Organization (UNESCO) meeting in Mexico City in 1997, the specific differentiation of the two morphologically identical species would seem relevant in clinical diagnosis. Several polymerase chain reaction (PCR)-based methods have been described and used successfully, but methods for DNA isolation from cysts in stool samples are time-consuming and problematic due to inhibitory factors in faeces. The use of the slightly modified QIAamp tissue method (Qiagen, Germany) for DNA isolation was evaluated in 657 unpreserved faecal samples from cases of suspected Entamoeba histolytica/Entamoeba dispar infection. In only 1.7% of the cases was PCR hampered by inhibitors present in the faeces. The DNA isolation procedure was found to be rapid, simple and one that could easily be implemented in a routine diagnostic setting. In 98.8% of Entamoeba histolytica/Entamoeba dispar cyst-positive faecal samples, the true identity of the cysts could be determined using PCR specific for Entamoeba histolytica and Entamoeba dispar, respectively.

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 of Clonorchis sinensis in stool samples using real-time PCR

Abstract Human clonorchiasis, caused by infection with the trematode Clonorchis sinensis, is a common health problem in East Asia. In an attempt to develop a new, sensitive method for the diagnosis of the disease, the use of a real-time PCR (targeting the internal-transcribed-spacer-2 sequence of the parasite) to detect C. sinensis-specific DNA in faecal samples has recently been evaluated. The PCR-based assay, which included an internal control to detect any inhibition of the amplification by faecal constituents in the sample, was performed on stool samples and on DNA controls representing a wide range of intestinal microorganisms. The assay appeared very specific, only showing positivity with C. sinensis and Opisthorchis felineus. The sensitivity of the assay was explored by testing 170 preselected samples of human faeces, from an endemic area of South Korea, which had known (microscopically-determined) densities of C. sinensis eggs. The sensitivity of the assay was 100% for the 74 samples that each had > 100 eggs/g and 91.4% for the other 70 samples found egg-positive by microcopy (i.e. those that had ≤ 100 eggs/g). Three of the 26 samples that appeared egg-negative by microscopy were found PCR-positive. Encouragingly, the PCR cycle-threshold values, which reflect parasite-specific DNA loads, showed significant correlation with the egg counts. The real-time PCR used in this study therefore appears to be a powerful tool for both the detection and quantification of C. sinensis infections.

PCR assay for the specific amplification of Oesophagostomum bifurcum DNA from human faeces

Oesophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodule worm) is of major human health significance in northern Togo and Ghana where the human hookworm, Necator americanus, also exists at high prevalence. Accurate diagnosis of O. bifurcum infection in humans is central to studying the epidemiology and controlling the parasite. To overcome limitations of current copro-diagnostic methods, we have developed an alternative, molecular approach. Utilising genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal DNA, we have established a two-step, semi-nested PCR method for the specific amplification of minute amounts (fg) of O. bifurcum DNA from human faecal samples. Using a panel of 155 well-defined faecal and DNA samples, the assay achieved a sensitivity of 94.6% and a specificity of 100%. This PCR assay will be useful for the diagnosis of O. bifurcum infection and as a molecular tool for elucidating the epidemiology of human oesophagostomiasis.

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.

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.