Comparison of real-time PCR and the Kato-Katz method for the diagnosis of soil-transmitted helminthiasis and assessment of cure in a randomized controlled trial

Diagnosis of soil-transmitted helminths (STHs) in developing countries is commonly based on microscopic detection of eggs in stool samples, using the Kato-Katz (KK) method, which has a poor sensitivity for detecting light intensity infections. We compared the performance of the KK method and real-time PCR in the framework of a randomized trial, which evaluated four novel treatments against Trichuris trichiura and concomitant STH infections.; Two stool samples obtained from 320 participants were examined at baseline and follow-up with quadruplicate KK and PCR analyses of one of the two samples using "bead-beating" for DNA extraction. At follow-up, 80 samples were negative according to both PCR and KK and 173 were positive with both methods for any of the STHs. Relative to PCR, the calculated sensitivity of KK at follow-up was 83.6%, 43.0% and 53.8% for T. trichiura, for hookworm and for Ascaris lumbricoides, respectively. The sensitivity of PCR compared with KK at this time point was 89.1% for T. trichiura, 72.7% for hookworm and 87.5% for A. lumbricoides. Cure rates (CRs) for T. trichiura and A. lumbricoides were slightly lower with the PCR method. For hookworm CRs with KK were mostly significantly lower, namely 36.7%, 91.1%, 72.2% and 77.8% for moxidectin, moxidectin in combination with tribendimidine, moxidectin in combination with albendazole and albendazole in combination with oxantel pamoate, respectively, whereas with PCR the CRs were 8.3%, 82.6%, 37.1% and 57.1%, respectively.; In conclusion, a single real-time PCR is as sensitive as quadruplicate KK for T. trichiura and A. lumbricoides detection but more sensitive for hookworm, which has an influence on the estimated treatment efficacy. PCR method with DNA extraction using the "bead-beating protocol" should be further promoted in endemic areas and laboratories that can afford the needed equipment. The study is registered at ISRCTN (no. 20398469)

Prevalence of intestinal protozoa infection among school-aged children on Pemba Island, Tanzania, and effect of single-dose albendazole, nitazoxanide and albendazole-nitazoxanide.

Pathogenic intestinal protozoa infections are common in school-aged children in the developing world and they are frequently associated with malabsorption syndromes and gastrointestinal morbidity. Since diagnosis of these parasites is difficult, prevalence data on intestinal protozoa is scarce. We collected two stool samples from school-aged children on Pemba Island, Tanzania, as part of a randomized controlled trial before and 3 weeks after treatment with (i) single-dose albendazole (400 mg); (ii) single-dose nitazoxanide (1,000 mg); (iii) nitazoxanide-albendazole combination (1,000 mg--400 mg), with each drug given separately on two consecutive days; and (iv) placebo. Formalin-fixed stool samples were examined for the presence of intestinal protozoa using an ether-concentration method to determine the prevalence and estimate cure rates (CRs). Almost half (48.7%) of the children were diagnosed with at least one of the (potentially) pathogenic protozoa Giardia intestinalis, Entamoeba histolytica/E. dispar and Blastocystis hominis. Observed CRs were high for all treatment arms, including placebo. Nitazoxanide showed a significant effect compared to placebo against the non-pathogenic protozoon Entamoeba coli. Intestinal protozoa infections might be of substantial health relevance even in settings where they are not considered as a health problem. Examination of a single stool sample with the ether-concentration method lacks sensitivity for the diagnosis of intestinal protozoa, and hence, care is indicated when interpreting prevalence estimates and treatment effects

Bacterial Populations in Complementary Foods and Drinking-water in Households with Children Aged 10-15 Months in Zanzibar, Tanzania

Bacteria were quantified in samples of drinking-water and in two porridges prepared for infant-feeding [fortified instant soy-rice porridge (SRP) and cooked porridge (Lishe bora, LB)] in 54 households. Bacterial numbers were measured again after the porridges had been held at room temperature for four hours (T4). Findings were benchmarked against bacterial numbers in traditional complementary foods sampled from 120 households. Total bacteria, coliform, and Enterobacteriaceae counts were enumerated using Petrifilm™. The mean log bacterial numbers were the lowest for LB at T0 (2.24±0.84 cfu/g aerobic counts) and the highest for SRP at T4 (4.63±0.56 cfu/g aerobic counts). The total bacteria, coliform and Enterobacteriaceae counts were higher at T4 than at T0 for LB (p≤0.001); however, only the coliform and Enterobacteriaceae counts were higher at T4 than at T0 for SRP (p<0.001). Drinking-water, SRP0, traditional foods, and SRP4 all had the mean aerobic counts higher than the acceptable cut-off but the total bacterial count in SRP0 was not significantly (p=0.543) different from drinking-water. However, coliform and Enterobacteriaceae counts in SRP0 were higher than in drinking-water (p<0.001). Also, although the aerobic counts of SRP4 were not significantly (p>0.999) different from traditional foods, the coliform and Enterobacteriaceae counts were significantly higher in SRP4 than in traditional foods (p<0.001). It is, therefore, recommended that food safety concerns be addressed when improving complementary foods

Assessment of anthelmintic efficacy of mebendazole in school children in six countries where soil-transmitted helminths are endemic

BACKGROUND: Robust reference values for fecal egg count reduction (FECR) rates of the most widely used anthelmintic drugs in preventive chemotherapy (PC) programs for controlling soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, and hookworm) are still lacking. However, they are urgently needed to ensure detection of reduced efficacies that are predicted to occur due to growing drug pressure. Here, using a standardized methodology, we assessed the FECR rate of a single oral dose of mebendazole (MEB; 500 mg) against STHs in six trials in school children in different locations around the world. Our results are compared with those previously obtained for similarly conducted trials of a single oral dose of albendazole (ALB; 400 mg). METHODOLOGY: The efficacy of MEB, as assessed by FECR, was determined in six trials involving 5,830 school children in Brazil, Cambodia, Cameroon, Ethiopia, United Republic of Tanzania, and Vietnam. The efficacy of MEB was compared to that of ALB as previously assessed in 8,841 school children in India and all the above-mentioned study sites, using identical methodologies. PRINCIPAL FINDINGS: The estimated FECR rate [95% confidence interval] of MEB was highest for A. lumbricoides (97.6% [95.8; 99.5]), followed by hookworm (79.6% [71.0; 88.3]). For T. trichiura, the estimated FECR rate was 63.1% [51.6; 74.6]. Compared to MEB, ALB was significantly more efficacious against hookworm (96.2% [91.1; 100], p CONCLUSIONS/SIGNIFICANCE: A minimum FECR rate of 95% for A. lumbricoides, 70% for hookworm, and 50% for T. trichiura is expected in MEB-dependent PC programs. Lower FECR results may indicate the development of potential drug resistance

Efficacy and Safety of Nitazoxanide, Albendazole, and Nitazoxanide-Albendazole against Trichuris trichiura Infection: A Randomized Controlled Trial

More than 5 billion people are at risk of infection with one of the three most common intestinal worms, the roundworm Ascaris lumbricoides, the whipworm Trichuris trichiura, and two different kinds of hookworms. The global strategy to control these intestinal worm infections is through the regular administration of deworming drugs to school-aged children (albendazole, 400 mg; mebendazole, 500 mg). However, especially against T. trichiura, a low treatment response is observed with single doses of both drugs. We tested the antiprotozoal drug nitazoxanide, which had shown promising trichuricidal properties in in vitro experiments. A randomized controlled trial was carried out on the island of Pemba in Tanzania. Four treatment arms were included: (i) single albendazole (400 mg), (ii) single nitazoxanide (1,000 mg), (iii) nitazoxanide-albendazole combination (1,000 mg–400 mg) with each drug given separately on two consecutive days, and (iv) placebo. Children were asked for adverse events at several time points after treatment. Nitazoxanide showed no ability to cure T. trichiura-infected children and caused significantly more mild adverse events than placebo. Albendazole and the nitazoxanide-albendazole combination showed only a minimal effect against T. trichiura. Our results emphasize the urgent need to develop new, safe, and effective anthelmintic drugs against T. trichiura

Dissection of the gut microbiota in mothers and children with chronic Trichuris trichiura infection in Pemba Island, Tanzania

Background: Soil-transmitted helminthiases are important neglected tropical diseases that result in a notably high number of disability-adjusted life years worldwide. Characterizing the interactions between the human intestinal microbiome and helminths is of interest in the development of alternative treatments that do not rely on chemotherapeutics and do not lead to drug resistance.MethodsWe recruited and obtained fecal samples from 32 pairs of mothers and children on Pemba Island and monitored their intestinal microbiota using 16S rRNA gene sequencing.ResultsWe observed that microbial changes occur in the gut microbiota of infected mothers and children. Some short-chain fatty acid (SCFA)-producing bacteria and carbohydrate-degrading bacteria exhibited lower abundance in the infected individuals. Potentially pathogenic Campylobacter and proinflammatory Methanobrevibacter in infected mothers and opportunistic Enterococcus in infected children exhibited greater abundance.ConclusionsOur findings could reveal the microbiota profiling in T. trichiura-infected individuals, indicate the potential roles of key microbiota in the host and aid to the development of novel strategies to control T. trichiura infection

Effi cacy of chlorhexidine application to umbilical cord on neonatal mortality in Pemba, Tanzania: a community-based randomised controlled trial

Background In low-income countries, including the east African region, a third of neonatal deaths are due to infections. A substantial proportion of these have been attributed to sepsis, which can result from umbilical cord infections. Evidence from Asia suggests that chlorhexidine application to the neonatal umbilical cord reduces mortality, but no data from Africa are available. We aimed to assess the eff ect of umbilical cord cleansing with 4% chlorhexidine solution on neonatal mortality and omphalitis in rural settings of sub-Saharan Africa. Methods We did a community-based randomised controlled trial on Pemba Island, Zanzibar, Tanzania. All eligible babies (aged 1 h to 48 h, without congenital malformations) from hospital-based and community-based deliveries on Pemba Island were enrolled. Participants were randomly assigned to either 4% free chlorhexidine for cord care or to dry cord care using a computer-generated random sequence. For babies allocated to the chlorhexidine group, mothers or caretakers were advised to apply the solution to the cord every day until 3 days after the cord had dropped off . Cord stumps were examined for redness, pus, swelling, and foul odour on day 0, 1, 4, 10, and 28. The primary outcome for this study was mortality until day 28 on an intention-to-treat basis. The trial is registered with ClinicalTrials.gov, number NCT01528852. Findings Between May 19, 2011, and Aug 31, 2014, 36 911 newborn babies were enrolled into the chlorhexidine (n=18 015) and dry cord care study (n=18 896) groups. 17 468 (96·9%) of 18 015 neonates in the chlorhexidine group were available for complete follow-up (28 days) compared with 18 384 (97·3%) of 18 896 neonates in the dry cord care group. Mortality rate in the chlorhexidine group (10·5 deaths per 1000 livebirths) was not signifi cantly lower than that in the dry cord care group (11·7 per 1000 livebirths; relative risk 0·90, 0·74–1·09; p=0·27). Interpretation Our fi ndings do not support the use of chlorhexidine for reduction of neonatal mortality in this east African setting, which might not justify a change in the WHO policy. To inform global policy, a detailed meta-analysis and pooled analysis needs to be undertaken using data from both African and Asian settings

The optimal timing of post-treatment sampling for the assessment of anthelminthic drug efficacy against Ascaris infections in humans

The egg reduction rate (ERR) is the current standard mean to assess the efficacy of drugs against human soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura and hookworm). Although the timing of post-treatment sampling is pivotal for a readily interpretation of drug efficacy, there is lack empirical data that allows recommending the optimal time point for a follow-up egg counting. In the present study, we re-analyzed both the kinetics of worm expulsion and egg output for Ascaris lumbricoides following a single oral dose of albendazole in a series of studies previously conducted in Kenyan communities. The results indicate that it takes up to 10 days post-treatment before the expulsion of both adult male and female Ascaris worms is completed, approximately 20% of the worms being expelled between day 7 and 10 post-treatment. The sequential analysis of the egg out put, indicated a poor ERR (89.4%) at day 7 post-treatment, but a 100% ERR at day 14 and 21 post-treatment. Based on our findings we recommend to wait at least 14 days after an albendazole treatment before conducting the follow-up egg count. Any sampling before this time point may result in biased ERR estimates, due the release of residual eggs from moribund or degenerating worms

The detection of Schistosoma bovis in livestock on Pemba Island, Zanzibar: a preliminary study

Schistosoma bovis is a parasitic trematode of ungulates transmitted by freshwater snails in Sub-Saharan Africa causing bovine intestinal schistosomiasis that leads to chronic morbidity and significant agricultural economic losses. The recently reported occurrence of Bulinus globosus infected with S. bovis for the first time on Pemba Island (Zanzibar, United Republic of Tanzania) is a cause of concern for livestock/wildlife health and complicates the surveillance of Schistosoma haematobium. To confirm that local cattle are infected with S. bovis, fresh faecal samples were collected from six adult cows surrounding two schistosomiasis transmission sites in Kinyasini, Pemba Island. Schistosome eggs were concentrated, egg hatching stimulated and miracidia were individually captured and identified by analysis of the partial mitochondrial cytochrome c oxidase subunit 1 (cox1) and the partial nuclear internal transcribed spacer region (ITS1+5.8S+ITS2). Two S. bovis miracidia were collected from one faecal sample with two cox1 haplotypes, one matching cox1 data obtained from S. bovis cercariae, collected previously at the same site in Pemba, the other matching S. bovis cox1 data originating from coastal Tanzania. The findings conclude that S. bovis transmission has been established on Pemba Island and is likely to have been imported through livestock trade with East Africa. Increasing the sensitivity of non-invasive diagnostics for bovine schistosomiasis, together with wider sampling, will enable a better assessment on the epidemiology of S. bovis on Pemba Island

Quantitative PCR in soil-transmitted helminth control programs : four potential applications

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