Performance of Microscopy for the Diagnosis of Malaria and Human African Trypanosomiasis by Diagnostic Laboratories in the Democratic Republic of the Congo: Results of a Nation-Wide External Quality Assessment

The present External Quality Assessment (EQA) assessed microscopy of blood parasites among diagnostic laboratories in the Democratic Republic of the Congo. The EQA addressed 445 participants in 10/11 provinces (October 2013–April 2014). Participants were sent a panel of five slides and asked to return a routinely stained slide which was assessed for quality of preparation and staining. Response rate was 89.9% (400/445). For slide 1 (no parasites), 30.6% participants reported malaria, mostly Plasmodium falciparum. Only 11.0% participants reported slide 2 (Plasmodium malariae) correctly, 71.0% reported “malaria” or “Plasmodium falciparum” (considered acceptable). Slide 3 contained Plasmodium falciparum (109/μl) and Trypanosoma brucei brucei trypomastigotes: they were each reported by 32.5% and 16.5% participants respectively, 6.0% reported both. Slide 4 (Trypanosoma) was recognised by 44.9% participants. Slide 5 (Plasmodium ovale) was correctly reported by 6.2% participants, another 68.8% replied “malaria” or “Plasmodium falciparum” (considered acceptable). Only 13.6% of routine slides returned were correctly prepared and stained. The proportion of correct/acceptable scores for at least 4/5 slides was higher among EQA-experienced participants compared to first time participants (40.9% versus 22.4%, p = 0.001) and higher among those being trained < 2 years ago compared to those who were not (42.9% versus 26.3%, p = 0.01). Among diagnostic laboratories in Democratic Republic of the Congo, performance of blood parasite microscopy including non-falciparum species and Trypanosoma was poor. Recent training and previous EQA participation were associated with a better performance.     

Environmental Drivers of Monkeypox Transmission in the Democratic Republic of the Congo

EcoHealth - Monkeypox (MPX) is an emergent severe zoonotic disease resembling that of smallpox. To date, most cases of human MPX have been reported in the Democratic Republic of the Congo (DRC)....     

Human Exposure to Wild Animals in the Sankuru Province of the Democratic Republic of the Congo

Due to the high level of biological diversity in the Congo Basin and human population dependence on bushmeat, the DRC represents an ideal location for expanding knowledge on wild animal exposures and thus the potential for transmission of zoonotic pathogens. However, limited information exists on patterns and extent of contact with wildlife in such communities. Using a cross-sectional study, 14 villages in the Sankuru Province of the DRC were surveyed between August and September 2007. Villagers ≥ 1 year of age and at home of the time of the survey were eligible and enrolled to describe and assess factors associated with animal exposures (both activity and type of animal). Among respondents, 91% reported exposure to rodents, 89% to duikers, 78% to non-human primates (NHPs), and 32% reported contact with bats in the month prior to the survey. The most frequently reported activities included eating (95%), cooking (70%), and butchering or skinning of animals (55%). The activities and animals to which subjects had contact varied by sex and age. Moreover, we observed a high correlation of the same activities across animal types. In this and other populations that rely on bushmeat, there is a high frequency of exposure to multiple animal species through various modalities. In the event of future zoonotic disease outbreaks, effective public health interventions and campaigns that mitigate the risk of animal contact during outbreaks need to be broad to include various modes of contact and should be directed to both men and women across all age groups. As available information is limited, further studies are necessary to better understand the complex relationships and exposures individuals have with animals.     

Simple Technique for in Field Samples Collection in the Cases of Skin Rash Illness and Subsequent PCR Detection of Orthopoxviruses and Varicella Zoster Virus

Background

In case of outbreak of rash illness in remote areas, clinically discriminating monkeypox (MPX) from severe form of chickenpox and from smallpox remains a concern for first responders.

Objective

The goal of the study was therefore to use MPX and chickenpox outbreaks in Democratic Republic of Congo (DRC) as a test case for establishing a rapid and specific diagnosis in affected remote areas.

Methods

In 2008 and 2009, successive outbreaks of presumed MPX skin rash were reported in Bena Tshiadi, Yangala and Ndesha healthcare districts of the West Kasai province (DRC). Specimens consisting of liquid vesicle dried on filter papers or crusted scabs from healing patients were sampled by first responders. A field analytical facility was deployed nearby in order to carry out a real-time PCR (qPCR) assay using genus consensus primers, consensus orthopoxvirus (OPV) and smallpox-specific probes spanning over the 14 kD fusion protein encoding gene. A PCR-restriction fragment length polymorphism was used on-site as backup method to confirm the presence of monkeypox virus (MPXV) in samples. To complete the differential diagnosis of skin rash, chickenpox was tested in parallel using a commercial qPCR assay. In a post-deployment step, a MPXV-specific pyrosequencing was carried out on all biotinylated amplicons generated on-site in order to confirm the on-site results.

Results

Whereas MPXV proved to be the agent causing the rash illness outbreak in the Bena Tshiadi, VZV was the causative agent of the disease in Yangala and Ndesha districts. In addition, each on-site result was later confirmed by MPXV-specific pyrosequencing analysis without any discrepancy.

Conclusion

This experience of rapid on-site dual use DNA-based differential diagnosis of rash illnesses demonstrates the potential of combining tests specifically identifying bioterrorism agents and agents causing natural outbreaks. This opens the way to rapid on-site DNA-based identification of a broad spectrum of causative agents in remote areas.     

Re-emergence of Crimean-Congo hemorrhagic fever virus in Central Africa

Background

Crimean-Congo hemorrhagic fever (CCHF) is a severe tick-borne disease well recognized through Europe and Asia where diagnostic tests and medical surveillance are available. However, it is largely neglected in Africa, especially in the tropical rainforest of Central Africa where only sporadic human cases have been reported and date back to more than 30 years. We describe here an isolated human case that occurred in the Democratic Republic of the Congo in 2008 and performed phylogenetic analysis to investigate whether it resulted from a regional re-emergence or from the introduction of a novel virus in the area.

Methods and Findings

Near complete segment S and partial segment M sequences were characterized. Bayesian phylogenetic analysis and datation were performed to investigate the relationship between this new strain and viral strains from Africa, Europe and Asia. The new strain is phylogenetically close to the previously described regional genotype (II) that appears to be specific to Central Africa. Phylogenetic discrepancy between segment S and M suggested genetic exchange among local sublineages, which was dated back to 130–590 years before present.

Conclusions

The phylogenetic analyses presented here suggest ongoing CCHF virus circulation in Central Africa for a long time despite the absence of reported human cases. Many infections have most probably been overlooked, due to the weakness of healthcare structures and the absence of available diagnostic procedure. However, despite the lack of accurate ecological data, the sporadic reporting of human cases could also be partly associated with a specific sylvatic cycle in Central Africa where deforestation may raise the risks of re-emergence. For these reasons, together with the high risk of nosocomial transmission, public health authorities'' attention should be drawn to this etiological agent.     

External Quality Assessment of Reading and Interpretation of Malaria Rapid Diagnostic Tests among 1849 End-Users in the Democratic Republic of the Congo through Short Message Service (SMS)

Background

Although malaria rapid diagnostic tests (RDT) are simple to perform, they remain subject to errors, mainly related to the post-analytical phase. We organized the first large scale SMS based external quality assessment (EQA) on correct reading and interpretation of photographs of a three-band malaria RDT among laboratory health workers in the Democratic Republic of the Congo (DR Congo).

Methods and Findings

High resolution EQA photographs of 10 RDT results together with a questionnaire were distributed to health facilities in 9 out of 11 provinces in DR Congo. Each laboratory health worker answered the EQA by Short Message Service (SMS). Filled-in questionnaires from each health facility were sent back to Kinshasa. A total of 1849 laboratory health workers in 1014 health facilities participated. Most frequent errors in RDT reading were i) failure to recognize invalid (13.2–32.5% ) or negative test results (9.8–12.8%), (ii) overlooking faint test lines (4.1–31.2%) and (iii) incorrect identification of the malaria species (12.1–17.4%). No uniform strategy for diagnosis of malaria at the health facility was present. Stock outs of RDTs occurred frequently. Half of the health facilities had not received an RDT training. Only two thirds used the RDT recommended by the National Malaria Control Program. Performance of RDT reading was positively associated with training and the technical level of health facility. Facilities with RDT positivity rates >50% and located in Eastern DR Congo performed worse.

Conclusions

Our study confirmed that errors in reading and interpretation of malaria RDTs are widespread and highlighted the problem of stock outs of RDTs. Adequate training of end-users in the application of malaria RDTs associated with regular EQAs is recommended.     

Pathogen-Host Associations and Predicted Range Shifts of Human Monkeypox in Response to Climate Change in Central Africa

Climate change is predicted to result in changes in the geographic ranges and local prevalence of infectious diseases, either through direct effects on the pathogen, or indirectly through range shifts in vector and reservoir species. To better understand the occurrence of monkeypox virus (MPXV), an emerging Orthopoxvirus in humans, under contemporary and future climate conditions, we used ecological niche modeling techniques in conjunction with climate and remote-sensing variables. We first created spatially explicit probability distributions of its candidate reservoir species in Africa''s Congo Basin. Reservoir species distributions were subsequently used to model current and projected future distributions of human monkeypox (MPX). Results indicate that forest clearing and climate are significant driving factors of the transmission of MPX from wildlife to humans under current climate conditions. Models under contemporary climate conditions performed well, as indicated by high values for the area under the receiver operator curve (AUC), and tests on spatially randomly and non-randomly omitted test data. Future projections were made on IPCC 4^th Assessment climate change scenarios for 2050 and 2080, ranging from more conservative to more aggressive, and representing the potential variation within which range shifts can be expected to occur. Future projections showed range shifts into regions where MPX has not been recorded previously. Increased suitability for MPX was predicted in eastern Democratic Republic of Congo. Models developed here are useful for identifying areas where environmental conditions may become more suitable for human MPX; targeting candidate reservoir species for future screening efforts; and prioritizing regions for future MPX surveillance efforts.     

Serum 8,12-iso-iPF2α-VI Isoprostane Marker of Oxidative Damage and Cognition Deficits in Children with Konzo

We sought to determine whether motor and cognitive deficits associated with cassava (food) cyanogenic poisoning were associated with high concentrations of F2-isoprostanes, well-established indicators of oxidative damage. Concentrations of serum F2-isoprostanes were quantified by LC-MS/MS and anchored to measures of motor proficiency and cognitive performance, which were respectively assessed through BOT-2 (Bruininks/Oseretsky Test, 2^nd Edition) and KABC-II (Kaufman Assessment Battery for Children, 2^nd edition) testing of 40 Congolese children (21 with konzo and 19 presumably healthy controls, overall mean age (SD): 9.3 (3.2) years). Exposure to cyanide was ascertained by concentrations of its main metabolite thiocyanate (SCN) in plasma and urine. Overall, SCN concentrations ranged from 91 to 325 and 172 to 1032 µmol/l in plasma and urine, respectively. Serum isoprostanes ranged from 0.1 to 0.8 (Isoprostane-III), 0.8 to 8.3 (total Isoprostane-III), 0.1 to 1.5 (Isoprostane-VI), 2.0 to 9.0 (total Isoprostane-VI), or 0.2 to 1.3 ng/ml (8,12-iso-iPF2α-VI isoprostane). Children with konzo poorly performed at the BOT-2 and KABC-II testing relative to presumably healthy children (p<0.01). Within regression models adjusting for age, gender, motor proficiency, and other biochemical variables, 8,12-iso-iPF2α-VI isoprostane was significantly associated with the overall cognitive performance (β = −32.36 (95% CI: −51.59 to −13.03; P<0.001). This model explained over 85% of variation of the KABC-II score in children with konzo, but was not significant in explaining the motor proficiency impairment. These findings suggest that cognitive deficits and, possibly, brain injury associated with cassava poisoning is mediated in part by oxidative damage in children with konzo. 8,12-iso-iPF2α-VI isoprostane appears to be a good marker of the neuropathogenic mechanisms of konzo and may be used to monitor the impact of interventional trials to prevent the neurotoxic effects of cassava cyanogenic poisoning.     

Multi Drug Resistant Tuberculosis in Mosango,a Rural Area in the Democratic Republic of Congo

Multidrug Resistant Tuberculosis (MDR-TB) is a serious threat which jeopardizes the worldwide efforts to control TB. The Democratic Republic of Congo (DRC) is one of 27 countries with a high burden of MDR-TB. Data on the magnitude, trends, and the distribution of MDR-TB in DRC are scanty. Kinshasa, the capital city of DRC which accounts for 20% of all TB cases nationwide, is notifying more than 80% of all MDR suspects. We report here a cluster of MDR-TB cases that was investigated in the Mosango health district, in the Bandundu south Province, DRC in 2008. Phenotypic Drug Sensitivity Testing and DNA sequencing were performed on 18 sputum specimens collected from 4 MDR-TB suspects and 5 household contacts. Sequencing data confirmed that the 4 suspects were indeed Rifampicin resistant cases. Sequencing of the rpoB gene showed that 3 cases (patients A, B and D) had a single mutation encoding a substitution to 526Tyr, 531Trp and 526Leu respectively. Patient C had a double mutation encoding a change to 531Leu and 633Leu. Two of the investigated cases died within 4 months of a second-line treatment course. Results highlight the need to enhance adequate laboratory services within the country for both clinical as well as surveillance purposes.