Stefanie Kramme

Diagnosing Schistosomiasis by Detection of Cell-Free Parasite DNA in Human Plasma

Introduction Schistosomiasis (bilharzia), one of the most relevant parasitoses of humans, is confirmed by microscopic detection of eggs in stool, urine, or organ biopsies. The sensitivity of these procedures is variable due to fluctuation of egg shedding. Serological tests on the other hand do not distinguish between active and past disease. In patients with acute disease (Katayama syndrome), both serology and direct detection may produce false negative results. To overcome these obstacles, we developed a novel diagnostic strategy, following the rationale that Schistosoma DNA may be liberated as a result of parasite turnover and reach the blood. Cell-free parasite DNA (CFPD) was detected in plasma by PCR. Methodology/Principal Findings Real-time PCR with internal control was developed and optimized for detection of CFPD in human plasma. Distribution was studied in a mouse model for Schistosoma replication and elimination, as well as in human patients seen before and after treatment. CFPD was detectable in mouse plasma, and its concentration correlated with the course of anti-Schistosoma treatment. Humans with chronic disease and eggs in stool or urine (n = 14) showed a 100% rate of CFPD detection. CFPD was also detected in all (n = 8) patients with Katayama syndrome. Patients in whom no viable eggs could be detected and who had been treated for schistomiasis in the past (n = 30) showed lower detection rates (33.3%) and significantly lower CFPD concentrations. The duration from treatment to total elimination of CFPD from plasma was projected to exceed one year. Conclusions/Significance PCR for detection of CFPD in human plasma may provide a new laboratory tool for diagnosing schistosomiasis in all phases of clinical disease, including the capacity to rule out Katayama syndrome and active disease. Further studies are needed to confirm the clinical usefulness of CFPD quantification in therapy monitoring.

Evaluation of Advanced Reverse Transcription-PCR Assays and an Alternative PCR Target Region for Detection of Severe Acute Respiratory Syndrome-Associated Coronavirus

ABSTRACT First-generation reverse transcription-PCR (RT-PCR) assays for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) gave false-negative results in a considerable fraction of patients. In the present study, we evaluated two second-generation, replicase (R) gene-based, real-time RT-PCR test kits—the RealArt HPA coronavirus LC kit (Artus, Hamburg, Germany) and the LightCycler SARS-CoV quantification kit (Roche, Penzberg, Germany)—and a real-time RT-PCR assay for the nucleocapsid (N) gene. Detecting the N-gene RNA might be advantageous due to its high abundance in cells. The kits achieved sensitivities of 70.8% (Artus) and 67.1% (Roche) in 66 specimens from patients with confirmed SARS (samples primarily from the upper and lower respiratory tract and stool). The sensitivity of the N-gene assay was 74.2%. The differences in all of the sensitivities were not statistically significant (P = 0.680 [analysis of variance]). Culture cells initially contained five times more N- than R-gene RNA, but the respective levels converged during 4 days of virus replication. In clinical samples the median concentrations of R- and N-gene RNA, respectively, were 1.2 × 106 and 2.8 × 106 copies/ml (sputum and endotracheal aspirates), 4.3 × 104 and 5.5 × 104 copies/ml (stool), and 5.5 × 102 and 5.2 × 102 copies/sample (throat swabs and saliva). Differences between the samples types were significant but not between the types of target RNA. All (n = 12) samples from the lower respiratory tract tested positive in all tests. In conclusion, the novel assays are more sensitive than the first-generation tests, but they still do not allow a comprehensive ruling out of SARS. Methods for the routine sampling of sputum without infection risk are needed to improve SARS RT-PCR.

Novel real-time PCR for the universal detection of Strongyloides species

Strongyloidiasis is a neglected disease that is prevalent mainly in tropical and subtropical regions. It is caused by intestinal nematodes of the genus Strongyloides. Due to the rise in worldwide travel, infections are increasingly encountered in non-endemic regions. Diagnosis is hampered by insensitive and laborious detection methods. A universal Strongyloides species real-time PCR was developed with an internal competitive control system. The 95% limit of detection as determined by probit analysis was one larva per PCR equivalent to 100 larvae per 200 mg stool. The assay proved to be 100% specific as assessed using a panel of parasites and bacteria and thus might be useful in the diagnostic setting as well as for Strongyloides research.

Detection and quantification of Mycobacterium leprae in tissue samples by real-time PCR

Real-time PCR technology has improved molecular diagnostics of many pathogens, but no such test is available for Mycobacterium leprae. In this report we describe the establishment and the pre-clinical evaluation of such an assay. The test achieved a theoretical analytical sensitivity limit of 194 M. leprae cells per skin biopsy specimen and facilitated quantification of mycobacteria in tissue over a range of 54–54,000,000 cells per sample. In punch skin biopsies from 39 untreated Ugandan patients with newly diagnosed leprosy, the clinical diagnosis could be confirmed in 88.9% of multibacillary and 33.3% of paucibacillary (microscopically negative) patients. Real-time detection thus did not increase the clinical sensitivity of PCR as compared to conventional protocols, in spite of its evidently high analytical sensitivity. On the other hand, as still no culture system exists for M. leprae, the assay appears to be a robust tool for detection of the bacterium in selected clinical situations, as well as for quantitation in experimental settings.

Orientia tsutsugamushi Bacteremia and Cytokine Levels in Vietnamese Scrub Typhus Patients

Scrub typhus, caused by the intracellular bacterium Orientia tsutsugamushi, is a major cause of febrile illness in the Asia/Pacific region. Here, we implemented a novel real-time PCR and determined the relation of DNA target gene concentration with serum cytokine levels. The limit of detection of the novel real-time PCR was 1,062 DNA copies per ml of EDTA whole blood. Specificity was excellent as determined on a panel of blood- and skin-borne bacteria, including Rickettsia spp. as well as healthy Vietnamese blood donors. Bacterial DNA concentrations after 9 to 12 days from symptoms onset were significantly higher than in earlier or later periods (P < 0.05). Significantly higher concentrations of gamma interferon (IFN-γ) and interleukin-10 (IL-10) occurred during the acute phase of disease (<10 days from onset) as opposed to the convalescent phase (P < 0.05). No significant differences were observed between the acute and the convalescent phases for tumor necrosis factor alpha (TNF-α) and IL-1β concentrations. Regression analysis of DNA concentrations and cytokine levels identified a significant positive relationship for IL-10 (P < 0.0182) but not for IFN-γ, TNF-α, and IL-1β. In conclusion, proinflammatory cytokines and IL-10 were differentially related to human bacteremia. They may thus be induced by different constituents of O. tsutsugamushi. As a future prospect in a clinical diagnostic laboratory, quantitative real-time PCR may serve as a reliable tool to monitor therapy and to detect treatment failure.

Detection of Mycobacterium tuberculosis by Real-Time PCR Using Pan-Mycobacterial Primers and a Pair of Fluorescence Resonance Energy Transfer Probes Specific for the M. tuberculosis Complex

PCR is widely used in clinical laboratories to diagnose pulmonary, extrapulmonary, and disseminated tuberculosis. A multitude of primer pairs have been successfully applied, one of which has been studied most extensively because it is included in the Roche Amplicor MTB assay, the only Food and Drug Administration-cleared PCR-based test for clinical detection of Mycobacterium tuberculosis (1)(2)(3). The test amplifies a 584-bp fragment of the 16s rDNA of all mycobacteria and identifies members of the M. tuberculosis complex (MTC) by hybridization of a specific DNA probe. However, the hybridization step extends the turnaround time of this test, and obligatory license fees render it unaffordable for application in experimental studies or resource-limited settings. Real-time detection technology has made it possible to establish noncommercial, probe-based PCR systems that provide stable operation, low contamination risk, and semiautomated interpretation of results (4)(5)(6)(7)(8)(9)(10)(11). We therefore aimed at adapting the Roche Amplicor assay to a real-time PCR protocol. We did not modify the primers of the Amplicor test because of their demonstrated performance; thus, only the detection probe was adapted to the requirements of real-time PCR. Classic real-time probes [5′-nuclease, “TaqMan” (12)] require cleavage by Taq polymerase (13), which could not be accomplished efficiently in our assay because the amplicon was too long and the only probe binding site specific for MTC was too distant from any of the primers (9)(12)(14). As an alternative approach, we chose a pair of fluorescence resonance energy transfer (FRET) probes that do not have to be cleaved, making them less dependent on the above-mentioned factors (15). On neighboring hybridization to the target DNA, an excited FRET probe system generates long-wavelength fluorescent emission that can be read by a Roche …

Q Fever in Young Children, Ghana

To the Editor: Recently, experts identified Q fever, caused by the small, gram-negative bacterium Coxiella burnetii, as an important underdiagnosed childhood disease (1). Studies on Q fever in children <5 years of age are scarce, especially with respect to sub-Saharan Africa. The only available study from Niger reports a seroprevalence of 9.6% (2). Throughout Africa, prevalence of Q fever in adults shows considerable variability and is highest in countries with prominent stockbreeding (3). Clinical manifestations of Q fever in children are similar to those of malaria (1,4). In malaria-endemic areas, most fevers are attributed to Plasmodium falciparum infection and presumptively treated with expensive combination therapies (5). In this context, other neglected fever-causing pathogens need to be given appropriate consideration. We studied the prevalence of Q fever antibodies in 219 randomly selected children living in 9 rural villages of the Ashanti region, Ghana. Plasma was obtained by venous puncture from 2-year-old children after they had participated in a malaria control study and had been clinically monitored for 21 months. Clinical, parasitologic, socioeconomic, and Global Positioning System information was recorded as described elsewhere (6,7). In addition, 158 healthy adult volunteers from the same area were included. Plasma was stored at –20°C until microimmunofluorescence assays (IFA) (Coxiella burnetii I+II, Vircell SL Microbiologists, Granada, Spain) were performed according to manufacturer’s instructions. To identify all children with Q fever titers, we regarded the following as positive fluorescence reactions to plasma dilutions: >1:64 for phase II immunoglobulin (Ig) G and >1:24 for phase II IgM with sensitivity (specificity) of 97.2% (100%) and 100% (56.3%), respectively. IgM testing was only performed on IgG-positive children. Positive and negative controls were run on each IFA slide. Relative risks (RR) for characteristics of children were calculated by χ2 test; p<0.05 was considered significant. Informed consent was obtained from all participants or their parents. The study protocol was approved by the committee on human research and publication, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. Positive C. burnetii phase II IgG responses were observed in 37 (16.9%) of 219 children and 14 (8.9%) of 158 adults (Figure, panels A and B). In comparison to adults, more children had IgG titers >64 (Figure, panels C and D). On the day of the serosurvey 71 (32.4%) of 219 children had fever (measured body temperature >38°C or reported fever within the previous 48 hours). Test outcome did not appear to be influenced by P. falciparum infection, since 4 of 37 IgG-positive children (23 of 182 IgG-negative children) had clinical malaria, 11/37 (62/182) had asymptomatic parasitemia, and 6/37 (38/182) had fever without parasitemia, and there were no significant differences between groups. The frequency of prior malaria episodes also did not influence antibody response. Three aparasitemic children had positive phase II IgM titers (24, 96, and 1,536; phase II IgG 64, 64, and 4,096, respectively). The child with the high IgM and IgG titers was clinically ill with nonsevere C. burnetii pneumonia. This child was among 10 (27%) of 37 phase II IgG-positive children with detectable anti–C. burnetii phase I antibodies. Of all sociodemographic characteristics under consideration, only maternal illiteracy was associated with positive phase II IgG testing (RR 2.1, 95% confidence interval 1.0–4.2, p<0.05). A considerable proportion of Ghanaian children had anti–C. burnetii antibodies, which indicates that Q fever might be a common event in this age group. Antibodies were more frequently detected in children than in adults. In adults, Q fever IgG antibodies reach a maximum 4–8 weeks after onset of symptoms and gradually decrease over months to finally fall below the detection limit (8). A long period since infection is less likely in young children, which could result in higher seropositivity. Children, especially those of illiterate mothers, could also be more frequently exposed to the pathogen. Consumption of unpasteurized dairy products can result in infection or seroconversion without clinical disease (9). However, because consumption of raw milk in the Ashanti region is regarded as being uncommon by local health authorities, we consider dairy products an unlikely source of the disease. Although participants were intensively exposed to P. falciparum, which causes polyclonal B-cell stimulation, malaria episodes and parasitemia with and without symptoms at time of the serosurvey did not influence testing (10). This finding is important because commercially available test kits have only been evaluated in Europeans not exposed to parasites. We cannot completely rule out the possibility that other infectious agents, which are either only prevalent or more prevalent in African populations, could have resulted in false-positive results. Nevertheless, the test method we used and existing data on cross-reactions weaken this hypothesis (8). We conclude that children in rural sub-Saharan Africa become exposed to C. burnetii early in life and that Q fever, which is clinically indistinguishable from malaria, may develop in an unknown proportion of them. The incidence of Q fever in relation to malaria, the route of infection, and appropriate serologic cutoffs for sub-Saharan Africa must be defined further. Currently, a prospective diagnostic study is investigating neglected infections, including human Q fever, as a cause of illness in Ghanaian children. Figure Seroprevalence of immunoglobulin (Ig) G antibodies against Coxiella burnetii phase II tested by microimmunofluorescence assays (IFA). A) Results of serologic tests of children, a cutoff titer of >64 for C. burnetii phase II IgG was applied; B) ...

High detection rate of Rickettsia africae in Amblyomma variegatum but low prevalence of anti-rickettsial antibodies in healthy pregnant women in Madagascar.

Tick-borne spotted fever group (SFG) rickettsioses are emerging infectious diseases in Sub-Saharan Africa. In Madagascar, the endemicity of tick-borne rickettsiae and their vectors has been incompletely studied. The first part of the present study was conducted in 2011 and 2012 to identify potential anthropophilic tick vectors for SFG rickettsiae on cattle from seven Malagasy regions, and to detect and characterize rickettsiae in these ticks. Amblyomma variegatum was the only anthropophilic tick species found on 262 cattle. Using a novel ompB-specific qPCR, screening for rickettsial DNA was performed on 111 A. variegatum ticks. Rickettsial DNA was detected in 96 of 111 ticks studied (86.5%). Rickettsia africae was identified as the only infecting rickettsia using phylogenetic analysis of ompA and ompB gene sequences and three variable intergenic spacers from 11 ticks. The second part of the study was a cross-sectional survey for antibodies against SFG rickettsiae in plasma samples taken from healthy, pregnant women at six locations in Madagascar, two at sea level and four between 450 and 1300m altitude. An indirect fluorescent antibody test with Rickettsia conorii as surrogate SFG rickettsial antigen was used. We found R. conorii-seropositives at all altitudes with prevalences between 0.5% and 3.1%. Our results suggest that A. variegatum ticks highly infected with R. africae are the most prevalent cattle-associated tick vectors for SFG rickettsiosis in Madagascar. Transmission of SFG rickettsiosis to humans occurs at different altitudes in Madagascar and should be considered as a relevant cause of febrile diseases.