The Trypanosoma cruzi Satellite DNA OligoC-TesT and Trypanosoma cruzi Kinetoplast DNA OligoC-TesT for Diagnosis of Chagas Disease: A Multi-cohort Comparative Evaluation Study

Background

The Trypanosoma cruzi satellite DNA (satDNA) OligoC-TesT is a standardised PCR format for diagnosis of Chagas disease. The sensitivity of the test is lower for discrete typing unit (DTU) TcI than for TcII-VI and the test has not been evaluated in chronic Chagas disease patients.

Methodology/Principal Findings

We developed a new prototype of the OligoC-TesT based on kinetoplast DNA (kDNA) detection. We evaluated the satDNA and kDNA OligoC-TesTs in a multi-cohort study with 187 chronic Chagas patients and 88 healthy endemic controls recruited in Argentina, Chile and Spain and 26 diseased non-endemic controls from D.R. Congo and Sudan. All specimens were tested in duplicate. The overall specificity in the controls was 99.1% (95% CI 95.2%–99.8%) for the satDNA OligoC-TesT and 97.4% (95% CI 92.6%–99.1%) for the kDNA OligoC-TesT. The overall sensitivity in the patients was 67.9% (95% CI 60.9%–74.2%) for the satDNA OligoC-TesT and 79.1% (95% CI 72.8%–84.4%) for the kDNA OligoC-Test.

Conclusions/Significance

Specificities of the two T. cruzi OligoC-TesT prototypes are high on non-endemic and endemic controls. Sensitivities are moderate but significantly (p = 0.0004) higher for the kDNA OligoC-TesT compared to the satDNA OligoC-TesT.     

Diagnostic Accuracy of the Leishmania OligoC-TesT and NASBA-Oligochromatography for Diagnosis of Leishmaniasis in Sudan

Background

The Leishmania OligoC-TesT and NASBA-Oligochromatography (OC) were recently developed for simplified and standardised molecular detection of Leishmania parasites in clinical specimens. We here present the phase II evaluation of both tests for diagnosis of visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and post kala-azar dermal leishmaniasis (PKDL) in Sudan.

Methodology

The diagnostic accuracy of the tests was evaluated on 90 confirmed and 90 suspected VL cases, 7 confirmed and 8 suspected CL cases, 2 confirmed PKDL cases and 50 healthy endemic controls from Gedarif state and Khartoum state in Sudan.

Principal Findings

The OligoC-TesT as well as the NASBA-OC showed a sensitivity of 96.8% (95% CI: 83.8%–99.4%) on lymph node aspirates and of 96.2% (95% CI: 89.4%–98.7%) on blood from the confirmed VL cases. The sensitivity on bone marrow was 96.9% (95% CI: 89.3%–99.1%) and 95.3% (95% CI: 87.1%–98.4%) for the OligoC-TesT and NASBA-OC, respectively. All confirmed CL and PKDL cases were positive with both tests. On the suspected VL cases, we observed a positive OligoC-TesT and NASBA-OC result in 37.1% (95% CI: 23.2%–53.7%) and 34.3% (95% CI: 20.8%–50.9%) on lymph, in 72.7% (95% CI: 55.8%–84.9%) and 63.6% (95% CI: 46.6%–77.8%) on bone marrow and in 76.9% (95% CI: 49.7%–91.8%) and 69.2% (95% CI: 42.4%–87.3%) on blood. Seven out of 8 CL suspected cases were positive with both tests. The specificity on the healthy endemic controls was 90% (95% CI: 78.6%–95.7%) for the OligoC-TesT and 100% (95% CI: 92.9%–100.0%) for the NASBA-OC test.

Conclusions

Both tests showed high sensitivity on lymph, blood and tissue scrapings for diagnosis of VL, CL and PKDL in Sudan, but the specificity for clinical VL was significantly higher with NASBA-OC.     

A history of chagas disease transmission, control, and re-emergence in peri-rural La Joya, Peru

Background

The history of Chagas disease control in Peru and many other nations is marked by scattered and poorly documented vector control campaigns. The complexities of human migration and sporadic control campaigns complicate evaluation of the burden of Chagas disease and dynamics of Trypanosoma cruzi transmission.

Methodology/Principal Findings

We conducted a cross-sectional serological and entomological study to evaluate temporal and spatial patterns of T. cruzi transmission in a peri-rural region of La Joya, Peru. We use a multivariate catalytic model and Bayesian methods to estimate incidence of infection over time and thereby elucidate the complex history of transmission in the area. Of 1,333 study participants, 101 (7.6%; 95% CI: 6.2–9.0%) were confirmed T. cruzi seropositive. Spatial clustering of parasitic infection was found in vector insects, but not in human cases. Expanded catalytic models suggest that transmission was interrupted in the study area in 1996 (95% credible interval: 1991–2000), with a resultant decline in the average annual incidence of infection from 0.9% (95% credible interval: 0.6–1.3%) to 0.1% (95% credible interval: 0.005–0.3%). Through a search of archival newspaper reports, we uncovered documentation of a 1995 vector control campaign, and thereby independently validated the model estimates.

Conclusions/Significance

High levels of T. cruzi transmission had been ongoing in peri-rural La Joya prior to interruption of parasite transmission through a little-documented vector control campaign in 1995. Despite the efficacy of the 1995 control campaign, T. cruzi was rapidly reemerging in vector populations in La Joya, emphasizing the need for continuing surveillance and control at the rural-urban interface.     

Prevalence,Clinical Staging and Risk for Blood-Borne Transmission of Chagas Disease among Latin American Migrants in Geneva,Switzerland

Background

Migration of Latin Americans to the USA, Canada and Europe has modified Chagas disease distribution, but data on imported cases and on risks of local transmission remain scarce. We assessed the prevalence and risk factors for Chagas disease, staged the disease and evaluated attitudes towards blood transfusion and organ transplant among Latin American migrants in Geneva, Switzerland.

Methodology/Principal Findings

This cross-sectional study included all consecutive Latin American migrants seeking medical care at a primary care facility or attending two Latino churches. After completing a questionnaire, they were screened for Chagas disease with two serological tests (Biomérieux ELISA cruzi; Biokit Bioelisa Chagas). Infected subjects underwent a complete medical work-up. Predictive factors for infection were assessed by univariate and multivariate logistic regression analysis.1012 persons (females: 83%; mean age: 37.2 [SD 11.3] years, Bolivians: 48% [n = 485]) were recruited. 96% had no residency permit. Chagas disease was diagnosed with two positive serological tests in 130 patients (12.8%; 95%CI 10.8%–14.9%), including 127 Bolivians (26.2%; 95%CI 22.3%–30.1%). All patients were in the chronic phase, including 11.3% with cardiac and 0.8% with digestive complications. Predictive factors for infection were Bolivian origin (OR 33.2; 95%CI 7.5–147.5), reported maternal infection with T. cruzi (OR 6.9; 95%CI 1.9–24.3), and age older than 35 years (OR 6.7; 95%CI 2.4–18.8). While 22 (16.9%) infected subjects had already donated blood, 24 (18.5%) and 34 (26.2%) considered donating blood and organs outside Latin America, respectively.

Conclusions

Chagas disease is highly prevalent among Bolivian migrants in Switzerland. Chronic cardiac and digestive complications were substantial. Screening of individuals at risk should be implemented in nonendemic countries and must include undocumented migrants.     

Use of a Novel Chagas Urine Nanoparticle Test (Chunap) for Diagnosis of Congenital Chagas Disease

Background

Detection of congenital T. cruzi transmission is considered one of the pillars of control programs of Chagas disease. Congenital transmission accounts for 25% of new infections with an estimated 15,000 infected infants per year. Current programs to detect congenital Chagas disease in Latin America utilize microscopy early in life and serology after 6 months. These programs suffer from low sensitivity by microscopy and high loss to follow-up later in infancy. We developed a Chagas urine nanoparticle test (Chunap) to concentrate, preserve and detect T. cruzi antigens in urine for early, non-invasive diagnosis of congenital Chagas disease.

Methodology/Principal Findings

This is a proof-of-concept study of Chunap for the early diagnosis of congenital Chagas disease. Poly N-isopropylacrylamide nano-particles functionalized with trypan blue were synthesized by precipitation polymerization and characterized with photon correlation spectroscopy. We evaluated the ability of the nanoparticles to capture, concentrate and preserve T. cruzi antigens. Urine samples from congenitally infected and uninfected infants were then concentrated using these nanoparticles. The antigens were eluted and detected by Western Blot using a monoclonal antibody against T. cruzi lipophosphoglycan. The nanoparticles concentrate T. cruzi antigens by 100 fold (western blot detection limit decreased from 50 ng/ml to 0.5 ng/ml). The sensitivity of Chunap in a single specimen at one month of age was 91.3% (21/23, 95% CI: 71.92%–98.68%), comparable to PCR in two specimens at 0 and 1 month (91.3%) and significantly higher than microscopy in two specimens (34.8%, 95% CI: 16.42%–57.26%). Chunap specificity was 96.5% (71/74 endemic, 12/12 non-endemic specimens). Particle-sequestered T. cruzi antigens were protected from trypsin digestion.

Conclusion/Significance

Chunap has the potential to be developed into a simple and sensitive test for the early diagnosis of congenital Chagas disease.     

High throughput selection of effective serodiagnostics for Trypanosoma cruzi infection

Background

Diagnosis of Trypanosoma cruzi infection by direct pathogen detection is complicated by the low parasite burden in subjects persistently infected with this agent of human Chagas disease. Determination of infection status by serological analysis has also been faulty, largely due to the lack of well-characterized parasite reagents for the detection of anti-parasite antibodies.

Methods

In this study, we screened more than 400 recombinant proteins of T. cruzi, including randomly selected and those known to be highly expressed in the parasite stages present in mammalian hosts, for the ability to detect anti-parasite antibodies in the sera of subjects with confirmed or suspected T. cruzi infection.

Findings

A set of 16 protein groups were identified and incorporated into a multiplex bead array format which detected 100% of >100 confirmed positive sera and also documented consistent, strong and broad responses in samples undetected or discordant using conventional serologic tests. Each serum had a distinct but highly stable reaction pattern. This diagnostic panel was also useful for monitoring drug treatment efficacy in chronic Chagas disease.

Conclusions

These results substantially extend the variety and quality of diagnostic targets for Chagas disease and offer a useful tool for determining treatment success or failure.     

Targeted screening strategies to detect Trypanosoma cruzi infection in children

Background

Millions of people are infected with Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. Anti-trypanosomal drug therapy can cure infected individuals, but treatment efficacy is highest early in infection. Vector control campaigns disrupt transmission of T. cruzi, but without timely diagnosis, children infected prior to vector control often miss the window of opportunity for effective chemotherapy.

Methods and Findings

We performed a serological survey in children 2–18 years old living in a peri-urban community of Arequipa, Peru, and linked the results to entomologic, spatial and census data gathered during a vector control campaign. 23 of 433 (5.3% [95% CI 3.4–7.9]) children were confirmed seropositive for T. cruzi infection by two methods. Spatial analysis revealed that households with infected children were very tightly clustered within looser clusters of households with parasite-infected vectors. Bayesian hierarchical mixed models, which controlled for clustering of infection, showed that a child''s risk of being seropositive increased by 20% per year of age and 4% per vector captured within the child''s house. Receiver operator characteristic (ROC) plots of best-fit models suggest that more than 83% of infected children could be identified while testing only 22% of eligible children.

Conclusions

We found evidence of spatially-focal vector-borne T. cruzi transmission in peri-urban Arequipa. Ongoing vector control campaigns, in addition to preventing further parasite transmission, facilitate the collection of data essential to identifying children at high risk of T. cruzi infection. Targeted screening strategies could make integration of diagnosis and treatment of children into Chagas disease control programs feasible in lower-resource settings.     

A Randomised Trial Evaluating the Safety and Immunogenicity of the Novel Single Oral Dose Typhoid Vaccine M01ZH09 in Healthy Vietnamese Children

Background

The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile.

Objectives

We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years.

Methods

Subjects were randomly assigned to receive either a nominal dose of 5×10⁹ CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA.

Principal Findings

One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18–5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12–36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI]  = 1.23 [0.550–2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 was detected in 51 (51%; 95% CI, 41–61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92–99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7–29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88–100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested.

Conclusions

This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children.

Trial Registration

Controlled-trials.comISRCTN91111837     

High Burden of Impetigo and Scabies in a Tropical Country

Background

Impetigo and scabies are endemic diseases in many tropical countries; however the epidemiology of these diseases is poorly understood in many areas, particularly in the Pacific.

Methodology/Principal Findings

We conducted three epidemiological studies in 2006 and 2007 to determine the burden of disease due to impetigo and scabies in children in Fiji using simple and easily reproducible methodology. Two studies were performed in primary school children (one study was a cross-sectional study and the other a prospective cohort study over ten months) and one study was performed in infants (cross-sectional). The prevalence of active impetigo was 25.6% (95% CI 24.1–27.1) in primary school children and 12.2% (95% CI 9.3–15.6) in infants. The prevalence of scabies was 18.5% (95% CI 17.2–19.8) in primary school children and 14.0% (95% CI 10.8–17.2) in infants. The incidence density of active impetigo, group A streptococcal (GAS) impetigo, Staphylococcus aureus impetigo and scabies was 122, 80, 64 and 51 cases per 100 child-years respectively. Impetigo was strongly associated with scabies infestation (odds ratio, OR, 2.4, 95% CI 1.6–3.7) and was more common in Indigenous Fijian children when compared with children of other ethnicities (OR 3.6, 95% CI 2.7–4.7). The majority of cases of active impetigo in the children in our study were caused by GAS. S. aureus was also a common cause (57.4% in school aged children and 69% in infants).

Conclusions/Significance

These data suggest that the impetigo and scabies disease burden in children in Fiji has been underestimated, and possibly other tropical developing countries in the Pacific. These diseases are more than benign nuisance diseases and consideration needs to be given to expanded public health initiatives to improve their control.     

Antigenicity and Diagnostic Potential of Vaccine Candidates in Human Chagas Disease

Background

Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease.

Methods and Results

Sera samples from inhabitants of the endemic areas of Argentina-Bolivia and Mexico-Guatemala were analyzed in 1^st-phase for anti-T. cruzi antibody response by traditional serology tests; and in 2^nd-phase for antibody response to the recombinant antigens (individually or mixed) by an ELISA. We noted similar antibody response to candidate antigens in sera samples from inhabitants of Argentina and Mexico (n = 175). The IgG antibodies to TcG1, TcG2, and TcG4 (individually) and TcG_mix were present in 62–71%, 65–78% and 72–82%, and 89–93% of the subjects, respectively, identified to be seropositive by traditional serology. Recombinant TcG1- (93.6%), TcG2- (96%), TcG4- (94.6%) and TcG_mix- (98%) based ELISA exhibited significantly higher specificity compared to that noted for T. cruzi trypomastigote-based ELISA (77.8%) in diagnosing T. cruzi-infection and avoiding cross-reactivity to Leishmania spp. No significant correlation was noted in the sera levels of antibody response and clinical severity of Chagas disease in seropositive subjects.

Conclusions

Three candidate antigens were recognized by antibody response in chagasic patients from two distinct study sites and expressed in diverse strains of the circulating parasites. A multiplex ELISA detecting antibody response to three antigens was highly sensitive and specific in diagnosing T. cruzi infection in humans, suggesting that a diagnostic kit based on TcG1, TcG2 and TcG4 recombinant proteins will be useful in diverse situations.     

Helminth Infection and Eosinophilia and the Risk of Plasmodium falciparum Malaria in 1- to 6-Year-Old Children in a Malaria Endemic Area

Background

Helminth infection is common in malaria endemic areas, and an interaction between the two would be of considerable public health importance. Animal models suggest that helminth infections may increase susceptibility to malaria, but epidemiological data has been limited and contradictory.

Methodology/Principal Findings

In a vaccine trial, we studied 387 one- to six-year-old children for the effect of helminth infections on febrile Plasmodium falciparum malaria episodes. Gastrointestinal helminth infection and eosinophilia were prevalent (25% and 50% respectively), but did not influence susceptibility to malaria. Hazard ratios were 1 for gastrointestinal helminth infection (95% CI 0.6–1.6) and 0.85 and 0.85 for mild and marked eosinophilia, respectively (95% CI 0.56–1.76 and 0.69–1.96). Incident rate ratios for multiple episodes were 0.83 for gastro-intestinal helminth infection (95% CI 0.5–1.33) and 0.86 and 0.98 for mild and marked eosinophilia (95% CI 0.5–1.4 and 0.6–1.5).

Conclusions/Significance

There was no evidence that infection with gastrointestinal helminths or urinary schistosomiasis increased susceptibility to Plasmodium falciparum malaria in this study. Larger studies including populations with a greater prevalence of helminth infection should be undertaken.     

Chagas disease among the Latin American adult population attending in a primary care center in Barcelona, Spain

Background/Aims

The epidemiology of Chagas disease, until recently confined to areas of continental Latin America, has undergone considerable changes in recent decades due to migration to other parts of the world, including Spain. We studied the prevalence of Chagas disease in Latin American patients treated at a health center in Barcelona and evaluated its clinical phase. We make some recommendations for screening for the disease.

Methodology/Principal Findings

We performed an observational, cross-sectional prevalence study by means of an immunochromatographic test screening of all continental Latin American patients over the age of 14 years visiting the health centre from October 2007 to October 2009. The diagnosis was confirmed by serological methods: conventional in-house ELISA (cELISA), a commercial kit (rELISA) and ELISA using T cruzi lysate (Ortho-Clinical Diagnostics) (oELISA). Of 766 patients studied, 22 were diagnosed with T. cruzi infection, showing a prevalence of 2.87% (95% CI, 1.6–4.12%). Of the infected patients, 45.45% men and 54.55% women, 21 were from Bolivia, showing a prevalence in the Bolivian subgroup (n = 127) of 16.53% (95% CI, 9.6–23.39%).All the infected patients were in a chronic phase of Chagas disease: 81% with the indeterminate form, 9.5% with the cardiac form and 9.5% with the cardiodigestive form. All patients infected with T. cruzi had heard of Chagas disease in their country of origin, 82% knew someone affected, and 77% had a significant history of living in adobe houses in rural areas.

Conclusions

We found a high prevalence of T. cruzi infection in immigrants from Bolivia. Detection of T. cruzi–infected persons by screening programs in non-endemic countries would control non-vectorial transmission and would benefit the persons affected, public health and national health systems.     

Real-time PCR in HIV/Trypanosoma cruzi coinfection with and without Chagas disease reactivation: association with HIV viral load and CD4 level

Background

Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed.

Methodology

Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not.

Principal Findings

qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman''s correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4⁺ cells or the CD4⁺/CD8⁺ ratio.

Conclusions

qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4⁺ cells/mm³ and the CD4⁺/CD8⁺ ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.     

Effect of Peer Health Workers on AIDS Care in Rakai,Uganda: A Cluster-Randomized Trial

Background

Human resource limitations are a challenge to the delivery of antiretroviral therapy (ART) in low-resource settings. We conducted a cluster randomized trial to assess the effect of community-based peer health workers (PHW) on AIDS care of adults in Rakai, Uganda.

Methodology/Principal Findings

15 AIDS clinics were randomized 2∶1 to receive the PHW intervention (n = 10) or control (n = 5). PHW tasks included clinic and home-based provision of counseling, clinical, adherence to ART, and social support. Primary outcomes were adherence and cumulative risk of virologic failure (>400 copies/mL). Secondary outcomes were virologic failure at each 24 week time point up to 192 weeks of ART. Analysis was by intention to treat. From May 2006 to July 2008, 1336 patients were followed. 444 (33%) of these patients were already on ART at the start of the study. No significant differences were found in lack of adherence (<95% pill count adherence risk ratio [RR] 0.55, 95% confidence interval [CI] 0.23–1.35; <100% adherence RR 1.10, 95% CI 0.94–1.30), cumulative risk of virologic failure (RR 0.81, 95% CI 0.61–1.08) or in shorter-term virologic outcomes (24 week virologic failure RR 0.93, 95% CI 0.65–1.32; 48 week, RR 0.83, 95% CI 0.47–1.48; 72 week, RR 0.81, 95% CI 0.44–1.49). However, virologic failure rates ≥96 weeks into ART were significantly decreased in the intervention arm compared to the control arm (96 week failure RR 0.50, 95% CI 0.31–0.81; 120 week, RR 0.59, 95% CI 0.22–1.60; 144 week, RR 0.39, 95% CI 0.16–0.95; 168 week, RR 0.30, 95% CI 0.097–0.92; 192 week, RR 0.067, 95% CI 0.0065–0.71).

Conclusions/Significance

A PHW intervention was associated with decreased virologic failure rates occurring 96 weeks and longer into ART, but did not affect cumulative risk of virologic failure, adherence measures, or shorter-term virologic outcomes. PHWs may be an effective intervention to sustain long-term ART in low-resource settings.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00675389","term_id":"NCT00675389"}}NCT00675389     

No Seasonal Accumulation of Resistant P. falciparum when High-Dose Chloroquine Is Used

Background

Potentially chloroquine resistant P. falciparum, identified by the 76T haplotype in the chloroquine resistance transporter (pfcrt 76T), are highly prevalent throughout Africa. In Guinea-Bissau, normal and double dose chloroquine have respective efficacies of 34% and 78% against P.falciparum with pfcrt 76T and approximately three times the normal dose of chloroquine is routinely taken. Proportions of pfcrt 76T generally increase during high transmission seasons, as P.falciparum with pfcrt 76T commonly survive treatment with normal dose chloroquine. In Guinea-Bissau, there should be no seasonal increase of pfcrt 76T if the high doses of CQ commonly used are effective.

Methods and Findings

P. falciparum parasite density, age, sex, the proportion of chloroquine resistance associated haplotypes pfcrt 76T and P. falciparum multidrug resistance gene 1 86Y were assessed in 988 samples collected from children between 2002 and 2007. There was no seasonal accumulation of any allele. During the high and low transmission periods the pfcrt 76T proportions were 24% (95% CI, 21–27%) and 26% (95% CI, 20–33%). There was no significant change of pfcrt 76T (OR 1.05, 95% CI; 0.94–1.16 p = 0.39) or pfmdr1 86Y (OR 0.92, 95%CI; 0.83–1.01 p = 0.08) proportions between 2003 and 2007. Lower median parasite density (P.falciparum/µl) was associated with pfcrt 76T (15254 [95% CI, 12737–17772]; n = 164) compared to pfcrt 76K (18664 [95% CI, 16676–20653]; p = 0.003; n = 591). Similarly, pfmdr1 86Y was associated with a lower median parasite density (16320 [95% CI, 13696–18944]; n = 224) compared to pfmdr1 86N, (18880 [95% CI, 16701–21059]; P = 0.018; n = 445).

Conclusions

In contrast to the rest of Africa, P. falciparum parasites resistant to normal dose chloroquine do not have a selective advantage great enough to become the dominant P.falciparum type in Guinea-Bissau. This is most likely due to the efficacy of high-dose chloroquine as used in Guinea-Bissau, combined with a loss of fitness associated with pfcrt 76T.     

Geohelminth Infections among Pregnant Women in Rural Western Kenya; a Cross-Sectional Study

Background

Geohelminth infections are common in rural western Kenya, but risk factors and effects among pregnant women are not clear.

Methodology

During a community-based cross-sectional survey, pregnant women were interviewed and asked to provide a blood sample and a single fecal sample. Hemoglobin was measured and a blood slide examined for malaria. Geohelminth infections were identified using the concentration and Kato-Katz method.

Results

Among 390 participants who provided a stool sample, 76.2% were infected with at least one geohelminth: 52.3% with Ascaris lumbricoides, 39.5% with hookworm, and 29.0% with Trichuris trichiura. Infection with at least one geohelminth species was associated with the use of an unprotected water source (adjusted odds ratio [AOR] 1.8, 95% confidence interval [CI] 1.1–3.0) and the lack of treatment of drinking water (AOR 1.8, 95% CI 1.1–3.1). Geohelminth infections were not associated with clinical symptoms, or low body mass index. A hookworm infection was associated with a lower mid upper arm circumference (adjusted mean decrease 0.7 cm, 95% CI 0.3–1.2 cm). Hookworm infections with an egg count ≥1000/gram feces (11 women) were associated with lower hemoglobin (adjusted mean decrease 1.5 g/dl, 95% CI 0.3–2.7). Among gravidae 2 and 3, women with A. lumbricoides were less likely to have malaria parasitemia (OR 0.4, 95% CI 0.2–0.8) compared to women without A. lumbricoides, unlike other gravidity groups.

Conclusion

Geohelminth infections are common in this pregnant population; however, there were few observed detrimental effects. Routine provision of antihelminth treatment during an antenatal clinic visit is recommended, but in this area an evaluation of the impact on pregnancy, malaria, and birth outcome is useful.     

Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis Using a Multiplex Real-Time PCR Assay

Background

Arduous to differ clinically, extrapulmonary tuberculosis and focal complications of brucellosis remain important causes of morbidity and mortality in many countries. We developed and applied a multiplex real-time PCR assay (M RT-PCR) for the simultaneous detection of Mycobacterium tuberculosis complex and Brucella spp.

Methodology

Conventional microbiological techniques and M RT-PCR for M. tuberculosis complex and Brucella spp were performed on 45 clinical specimens from patients with focal complications of brucellosis or extrapulmonary tuberculosis and 26 control samples. Fragments of 207 bp and 164 bp from the conserved region of the genes coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and the intergenic region SenX3-RegX3 were used for the identification of Brucella and M. tuberculosis complex, respectively.

Conclusions

The detection limit of the M RT-PCR was 2 genomes per reaction for both pathogens and the intra- and inter-assay coefficients of variation were 0.44% and 0.93% for Brucella and 0.58% and 1.12% for Mycobacterium. M RT-PCR correctly identified 42 of the 45 samples from patients with tuberculosis or brucellosis and was negative in all the controls. Thus, the overall sensitivity, specificity, PPV and NPV values of the M RT PCR assay were 93.3%, 100%, 100% and 89.7%, respectively, with an accuracy of 95.8% (95% CI, 91.1%–100%). Since M RT-PCR is highly reproducible and more rapid and sensitive than conventional microbiological tests, this technique could be a promising and practical approach for the differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.     

CD40: Novel Association with Crohn's Disease and Replication in Multiple Sclerosis Susceptibility

Background

A functional polymorphism located at −1 from the start codon of the CD40 gene, rs1883832, was previously reported to disrupt a Kozak sequence essential for translation. It has been consistently associated with Graves'' disease risk in populations of different ethnicity and genetic proxies of this variant evaluated in genome-wide association studies have shown evidence of an effect in rheumatoid arthritis and multiple sclerosis (MS) susceptibility. However, the protective allele associated with Graves'' disease or rheumatoid arthritis has shown a risk role in MS, an effect that we aimed to replicate in the present work. We hypothesized that this functional polymorphism might also show an association with other complex autoimmune condition such as inflammatory bowel disease, given the CD40 overexpression previously observed in Crohn''s disease (CD) lesions.

Methodology

Genotyping of rs1883832C>T was performed in 1564 MS, 1102 CD and 969 ulcerative colitis (UC) Spanish patients and in 2948 ethnically matched controls by TaqMan chemistry.

Principal Findings

The observed effect of the minor allele rs1883832T was replicated in our independent Spanish MS cohort [p = 0.025; OR (95% CI) = 1.12 (1.01–1.23)]. The frequency of the minor allele was also significantly higher in CD patients than in controls [p = 0.002; OR (95% CI) = 1.19 (1.06–1.33)]. This increased predisposition was not detected in UC patients [p = 0.5; OR (95% CI) = 1.04 (0.93–1.17)].

Conclusion

The impact of CD40 rs1883832 on MS and CD risk points to a common signaling shared by these autoimmune conditions.     

Antibody-Based Detection Tests for the Diagnosis of Helicobacter pylori Infection in Children: A Meta-Analysis

Background

Numerous serologic tests are available for the diagnosis of H. pylori infection in children. Common designs of antibody-based detection tests are ELISA and Western Blot (WB). For developing countries with limited laboratory resources and access, ELISA would be the preferred method because of its simplicity, lower cost and speed. Although in adults ELISA has proven to be highly accurate in diagnosing H. pylori infection; in children, it has shown variable accuracy.

Methods/Findings

We conducted a systematic review and meta-analysis to assess the accuracy of antibody-based detection tests for the diagnosis of H. pylori infection in children. Selection criteria included participation of at least 30 children and the use of a gold standard for H. pylori diagnosis. In a comprehensive search we identified 68 studies. Subgroup analyses were carried out by technique, immunoglobulin class, and source of test (commercial and in-house). The results demonstrated: 1) WB tests showed high overall performance, sensitivity 91.3% (95% CI, 88.9–93.3), specificity 89% (95% CI, 85.7–91.9), LR+ 8.2 (95% CI, 5.1–13.3), LR− 0.06 (95% CI, 0.02–0.16), DOR 158.8 (95% CI, 57.8–435.8); 2) ELISA-IgG assays showed low sensitivity 79.2% (95% CI, 77.3–81.0) and high specificity (92.4%, 95% CI, 91.6–93.3); 3) ELISA commercial tests varied widely in performance (test for heterogeneity p<0.0001); and 4) In-house ELISA with whole-cell antigen tests showed the highest overall performance: sensitivity 94% (95% CI, 90.2–96.7), specificity 96.4% (95% CI, 94.2–97.9), LR+ 19.9 (95% CI, 7.9–49.8), LR− 0.08 (95% CI, 0.04–0.15) DOR 292.8 (95% CI, 101.8–841.7).

Conclusions/Significance

WB test and in-house ELISA with whole-cell antigen tests are the most reliable tests for the diagnosis of H. pylori infection in children. Antigens obtained from local strains of the community could partially explain the good overall accuracy of the in-house ELISA. Because of its cost and technical demands, in-house ELISA might be more suitable for use in developing countries.