Multiplex-RT-PCR-ELISA panel for detecting mosquito-borne pathogens: Plasmodium sp. preserved and eluted from dried blood spots on sample cards.

BACKGROUND: Children are the most vulnerable group affected by malaria and other tropical, vector-borne diseases in low-resource countries. Infants presenting with acute onset fever represent a major sector of outpatient care in the Lake Victoria region. Misclassification and overuse of antibiotics and anti-malarial medications are consistent problems. Identifying the prevalent mosquito-borne pathogens in the region will reduce the prescription of non-indicated medicines. METHODS: The literature was reviewed focusing on the mosquito-borne pathogens most prevalent in sub-Saharan Africa. Accordingly, an assay comprised of a multiplex-reverse transcriptase-polymerase chain reaction and an enzyme-linked immunosorbent assay (multiplex-RT-PCR-ELISA) was designed and validated in its ability to identify and differentiate nine human mosquito-borne pathogens including eight arboviruses and Plasmodium sp., the aetiologic agents of malaria. Blood samples obtained from 132 children suspected of having malaria were spotted and preserved on Whatman® 903 protein sample cards. Multiplex-RT-PCR-ELISA analysis was assessed and compared to results obtained by blood smear microscopy and the malaria rapid diagnostic test (RDT). RESULTS: Nine out of nine pathogens were amplified specifically by the multiplex-RT-PCR-ELISA panel. Twenty-seven out of 132 paediatric patients presenting with acute fever were infected with Plasmodium sp., confirmed by multiplex-RT-PCR. The results of blood smear microscopy were only 40% sensitive and 92.8% specific. The malaria RDT, on the other hand, detected acute Plasmodium infections with 96.3% sensitivity and 98.1% specificity. The preservation of Plasmodium sp. in clinical sera and whole blood samples spotted on sample cards was evaluated. The duration of successful, sample card storage was 186 to 312 days. CONCLUSIONS: Reliable, easy-to-use point of care diagnostic tests are a powerful alternative to laboratory-dependent gold standard tests. The multiplex-RT-PCR-ELISA amplified and identified nine vector-borne pathogens including Plasmodium sp. with great accuracy. Translation of improved diagnostic approaches, i.e., multiplex-RT-PCR-ELISA, into effective treatment options promises to reduce childhood mortality and non-indicated prescriptions.

Extensive Antibiotic and Antimalarial Prescription Rate among Children with Acute Febrile Diseases in the Lake Victoria Region, Tanzania.

OBJECTIVES: Acute mosquito-borne febrile diseases pose a threat to children in the Sub-Saharan-Africa with ∼272 000 children dying worldwide from malaria in 2018. Although the awareness for malaria in this area has increased due to improved health education, the apparent decline of actual malaria cases has not affected clinical practice significantly. This study collected clinical and epidemiologic data of children presenting with acute febrile diseases in order delineate their diagnostic and therapeutic management. METHODS: A hospital-based cross-sectional clinical study was conducted at the Sekou Toure Regional Referral Hospital in Tanzania. Children between 1 month and 12 years of age with an axillary temperature ≥ 37.5°C were recruited from August 2016 to December 2016. Children received full clinical examination. In addition, file data about diagnostics and treatment were collected and malaria rapid diagnostic tests (mRDTs) were performed. Confirmatory malaria polymerase chain reaction was performed from dry blood spots. RESULTS: From 1381 children presented in the pediatric outpatient department, 133 met the inclusion criteria. Out of 133 febrile children, 10.5% were malaria positive. Treatment data indicate the prescription of antimalarials in 35.3% and antibiotics in 63.9% of the children with an overlap of 24.1% receiving both. Despite a negative mRDT, 36 patients received antimalarials. CONCLUSIONS: The findings of this study confirm a significant decline of malaria cases in the Lake Victoria region. The discrepancy between the valuable results provided by mRDTs and the high prescription rates of antibiotics and antimalarials call for an enforced diagnostic and therapeutic algorithm. LAY SUMMARY: The aim of the study was to take a closer look at reported cases of febrile diseases in the Lake Victoria region and assess the relationship between clinical as well as diagnostic findings and the resulting therapeutic concept. Based on these findings the prescription rate of antimalarial and antibiotic drugs was analyzed. The results showed an overall high prescription rate of antimalarials and antibiotics in both diagnosed malaria cases and cases with diagnosed bacterial infections.Not only with regards to the possible side effects of these medications but also keeping in mind the apparent misuse of resources this practice poses a serious burden to the health care system in this low resource country.

Challenges in Diagnosing and Treating Acutely Febrile Children with Suspected Malaria at Health Care Facilities in the Lake Mwanza Region of Tanzania.

Acute febrile diseases transmitted by mosquitos are a diagnostic challenge for pediatricians working in sub-Saharan Africa. Misclassification due to the lack of rapid, reliable diagnostic tests leads to the overuse of antibiotics and antimalarials. Children presenting with acute fever and suspected of having malaria were examined at health care facilities in the Mwanza Region of Tanzania. The sensitivity and specificity of blood smear microscopy and malaria rapid diagnostic tests that targeted histidine-rich protein 2 and Plasmodium lactate dehydrogenase were compared with a multiplex reverse transcriptase-polymerase chain reaction (PCR)-ELISA. Six hundred ninety-eight children presented with acute fever and met the criteria for inclusion; 23% received antibiotics and 23% received antimalarials prior to admission. Subsequently, 20% were confirmed by PCR to have Plasmodium falciparum infection. Blood smear microscopy exhibited 33% sensitivity and 93% specificity. The malaria rapid test provided 87% sensitivity and 98% specificity in detecting acute malaria infections. Only 7% of malaria-negative children received antimalarials at Sengerema Designated District Hospital when treatment was guided by the results of rapid testing. In contrast, 75% of malaria-negative patients were treated with antimalarial drugs at health facilities that used blood smears as the standard diagnostic test. Misclassification and premedication of nonmalarial, febrile illnesses contribute to the emergence of antimalarial and antimicrobial resistance. The incorporation of malaria rapid diagnostic tests into the clinical routine translated into improved treatment and a significant reduction in antimalarial drug prescriptions.

Multiplex Reverse Transcription Polymerase Chain Reaction Combined with a Microwell Hybridization Assay Screening for Arbovirus and Parasitic Infections in Febrile Patients Living in Endemic Regions of Colombia.

Acute febrile syndrome is a frequent reason for medical consultations in tropical and subtropical countries where the cause could have an infectious origin. Malaria and dengue are the primary etiologies in Colombia. As such, constant epidemiological surveillance and new diagnostic tools are required to identify the causative agents. A descriptive cross-sectional study was conducted to evaluate the circulation and differential diagnosis of six pathogens in two regions of Colombia. The results obtained via multiplex reverse transcription polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR-ELISA) were comparable to those obtained using rapid tests conducted at the time of patient enrollment. Of 155 patients evaluated, 25 (16.1%) and 16 (10.3%) were positive for malaria and dengue, respectively; no samples were positive for any of the other infectious agents tested. In most cases, m-RT-PCR-ELISA confirmed the results previously obtained through rapid testing.

Seroprevalence of Dengue and Chikungunya Virus Infections in Children Living in Sub-Saharan Africa: Systematic Review and Meta-Analysis.

Dengue and chikungunya viruses are frequent causes of malarial-like febrile illness in children. The rapid increase in virus transmission by mosquitoes is a global health concern. This is the first systematic review and meta-analysis of the childhood prevalence of dengue and chikungunya in Sub-Saharan Africa (SSA). A comprehensive search of the MEDLINE (Ovid), Embase (Ovid), and Cochrane Library (Wiley) databases was conducted on 28 June 2019, and updated on 12 February 2022. The search strategy was designed to retrieve all articles pertaining to arboviruses in SSA children using both controlled vocabulary and keywords. The pooled (weighted) proportion of dengue and chikungunya was estimated using a random effect model. The overall pooled prevalence of dengue and chikungunya in SSA children was estimated to be 16% and 7%, respectively. Prevalence was slightly lower during the period 2010-2020 compared to 2000-2009. The study design varied depending on the healthcare facility reporting the disease outbreak. Importantly, laboratory methods used to detect arbovirus infections differed. The present review documents the prevalence of dengue and chikungunya in pediatric patients throughout SSA. The results provide unprecedented insight into the transmission of dengue and chikungunya viruses among these children and highlight the need for enhanced surveillance and controlled methodology.