Morphological, molecular, and MALDI-TOF MS identification of bed bugs and associated Wolbachia species from Cameroon.

After vanishing from the public eye for more than 50 years, bed bugs have resurged to become one of the most widely discussed and heavily researched insect pests in the world. This study presents the basic information of infestations of tropical bed bugs, Cimex hemipterus (Hemiptera: Cimicidae), in Cameroon. A total of 248 immature stage and adult bed bug specimens were collected from households and a travel agency in Yaoundé and Douala, Cameroon. The ability of MALDI-TOF MS to identify bed bugs was tested using heads for adults and cephalothoraxes for immature stages. Microorganism screening was performed by qPCR and confirmed by regular PCR and sequencing. Based on morphometrical criteria, four stages of immature bed bugs are represented. Of the 248 bed bug specimens morphologically identified as Cimex hemipterus, 246 (77 males, 65 females and 104 immature specimens) were submitted to MALDI-TOF MS analysis. Of the 222 adults and immature specimens tested, 122 (59.9 %) produced good quality MALDI-TOF MS spectra (35 adults and 87 immature specimens). Blind testing allowed species level identification of 98.21 % of adult and immature C. hemipterus. Among the bacteria tested, only Wolbachia DNA was found in 12/246 (4.8 %) bed bugs. More surveys in the country are warranted to assess the true level of bed bug infestations, in order to take appropriate action for their control.

MALDI-TOF MS identification of cattle ticks from Cameroon.

MALDI-TOF MS has recently been proposed as an accurate tool for arthropod identification, including ticks. In this study, we evaluate and confirm the ability of MALDI-TOF MS, to identify different tick species collected in Cameroon, considering other lines of evidence (morphology and molecular). A total of 1483 adult ticks were collected from cattle in five distinct sites in the Western Highland of Cameroon. Because of engorged status and/or absence of some morphological criteria, some Ixodes spp. and Rhipicephalus spp. were identified to the genus level only. Among those, 944 ticks (543 males and 401 females) were selected for the current work. They were classified into 5 genera and 11 species: Rhipicephalus (Boophilus) microplus (31.7%), Rhipicephalus lunulatus (26%), Amblyomma variegatum (23%), Rhipicephalus sanguineus s.l. (4.8%), of Haemaphysalis leachi group (4.6%), Hyalomma truncatum (2.6%), Hyalomma rufipes (1.7%), Rhipicephalus muhsamae (1.1%), Rhipicephalus (Boophilus) annulatus (0.6%), Rhipicephalus (Boophilus) decoloratus (0.3%), Ixodes rasus (0.1%), Ixodes spp. (0.2%) and Rhipicephalus spp. (3.3%). Tick legs were subjected to MALDI-TOF MS analyzes, and the spectra of 929 (98.4%) specimens were of good quality. Analysis of these spectra provided intra-species reproducibility and interspecies specificity of MS profiles obtained from the different species. Our in-house MALDI-TOF MS arthropod database was upgraded with spectra from 44 specimens of 10 different tick species. Blind testing of good quality spectra revealed that 99% agreed with the morphological identification. Of these, 96.9% had log score values (LSVs) between 1.73 and 2.57. MALDI-TOF MS also allowed to correct the morphological misidentification of 7 ticks, and to identify 32 engorged ticks that were not morphologically identifiable to the species level. This study supports MALDI-TOF MS as a reliable tool for tick identification and provides new data on tick species identification in Cameroon.

High prevalence of asymptomatic Plasmodium falciparum malaria in Makenene, a locality in the forest-savannah transition zone, Centre Region of Cameroon.

Malaria transmission and prevalence is still not well documented across Cameroon particularly in medium-sized cities or localities representing high transit zone. Different risk factors could be associated with persistence malaria transmission such as population movement from high to low transmission settings. A cross-sectional community-based study was carried out to determine malaria prevalence and risk factors in Makenene, a small city in a forest-savannah which is a crossroads between different parts of the country where travellers usually stop-over day and night to rest. Using malaria diagnostic test (mRDTs from SD-BIOLINE) and microscopy (thin and thick blood smears), 406 participants from 237 households were tested for malaria infection. The prevalence of malaria was high irrespective of the detection method: mRDT (41.87%) or microscopy (38.42%). At household level, 46.41% of households had at least one case of malaria with an average of 1.41 infected individuals per household. Parasite density was also high with the majority of infected individuals (64.74%) bearing more than 500 parasites/µl. Only Plasmodium falciparum was found. The chances of being infected with malaria parasites was almost the same for all participants irrespective of the sleeping behavior, bednet usage, house type and environmental factors. The study supports high malaria transmission in the locality and the need for additional studies on vectors bionomics and transmission patterns.

Detection of Tick-Borne Pathogens in Ticks from Cattle in Western Highlands of Cameroon.

This study aimed to detect and identify microorganisms in ticks collected in the Western Highlands of Cameroon. Quantitative real-time and standard PCR assays, coupled with sequencing, were used. A total of 944 ticks collected from cattle in five distinct sites in Cameroon were selected for the analyses. They belonged to five genera (Amblyomma, Hyalomma, Rhipicephalus, Haemaphysalis, and Ixodes) and twelve species. Real-time PCR revealed that 23% (n = 218) of the ticks were positive for Rickettsia spp., 15% (n = 141) for bacteria of the Anaplasmataceae family, 3% (n = 29) for Piroplasmida, 0.5% (n = 5) for Coxiella burnetii, 0.4% (n = 4) for Borrelia spp., and 0.2% (n = 2) for Bartonella spp. The co-infection rate (3.4%, n = 32) involved mainly Rickettsia spp. and Anaplasmataceae. Of the Rickettsia spp. positive ticks, the targeted PCR and sequencing yielded Rickettsia africae (78.9%), Rickettsia aeschlimannii (6.4%), Rickettsia massiliae (7.8%), Candidatus Rickettsia barbariae (0.9%), and Rickettsia sp. (0.9%). Anaplasmataceae included Anaplasma marginale (4.3%), Anaplasma platys (1.4%), Anaplasma centrale (0.7%), Ehrlichia ruminantium (0.7%), Wolbachia sp., Candidatus Ehrlichia rustica (13.5%), Candidatus Ehrlichia urmitei (7%), and an uncultured Ehrlichia sp. (4.2%). Borrelia theileri was identified in one Rhipicephalus microplus tick. Unfortunately, Piroplasmida could not be identified to the species level. This study demonstrates that in Cameroon, ticks harbour a wide variety of microorganisms and present a risk of zoonotic diseases.

Performance assessment of a widely used rapid diagnostic test CareStart™ compared to microscopy for the detection of Plasmodium in asymptomatic patients in the Western region of Cameroon.

While malaria remains a serious public health concern, its rapid or prompt diagnosis in remote areas is important in the fight against the disease. The study aimed to evaluate the performance of widely used Rapid Diagnostic Test (RDT) kits for routinely detection of Plasmodium asymptomatic patients. A total of 400 asymptomatic participants of both sexes aged between 1-89 years from Menoua Division (Santchou and Dschang) were tested for malaria infection using both microscopy and CareStart™ RDT. The prevalence of malaria was higher when using the standard gold tool (Microscopy) (26.0%) compared to RDT (21.8%) with a significant difference (P < 0.05). However, a strong agreement was observed between both tests (kappa = 0.883; P < 0.0001). RDT CareStart™ sensitivity and specificity were 83.65% and 100% respectively while the positive predictive value and negative predictive value were 100% and 95.57% respectively. RDT sensitivity increased with parasite density while false negative (40.4%; n = 17) were observed only when parasite density was low (<500 parasites per microliter of blood). RDT kits appear to be good tools in areas where malaria diagnosis through microscopy is not feasible. However, the low sensibility observed when parasite density is low could be a concern.