A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer

Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3′ untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n = 220) and individuals not suspected of dengue virus infection (n = 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries

Author correction : Divergence across mitochondrial genomes of sympatric members of the 'Schistosoma indicum' group and clues into the evolution of 'Schistosoma spindale'

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Revisiting clinico-epidemiological pattern of human rickettsial infections in the central region of Sri Lanka: a hospital based descriptive study

Abstract Background This study revisits the clinico-epidemiology and serological patterns of rickettsioses in the central region of Sri Lanka and highlights the need of advanced diagnostics for precise identification of species responsible for rickettsioses. Methods The patients treated for rickettsioses between November 2009 and October 2011 were recruited for the study from Teaching Hospital, Peradeniya. Clinical characteristics and serology results were used for diagnosis. Results Study included 210 patients (mean age 44 years ± 3.2) and of them 188 (90%) had positive IgG and/or IgM sero-reactivity for spotted fever group (SFG). Of them, 134 had IgG titre ≥1/256 for SFG and presented with fever and skin rash. They also had headache [n = 119 (89%)], myalgia [n = 103 (77%)], arthralgia [n = 89 (66%)] of large joints, conjunctival injections [n = 83 (62%)], thrombocytopenia (n = 78.58%), anaemia (n = 14.10%), leukocytosis [n = 35 (26%)], leucopenia [n = 17 (13%)], elevated aspartate transaminase [n = 69 (52%)] and alanine transaminase [n = 73 (55%)]. Conclusions Predominance of SFG rickettsioses are reiterated, possibly transmitted by ticks. Joint disease is common with occasional fern leaf skin necrosis. Changing socio-economic conditions, vegetations, contact with domestic and wild animals, abundance of vectors would have contributed for emergence and sustenance of SFG in the region. Further research is needed to identify the causative agents and the mode of transmission

Prevalence and Carrier Status of Leptospirosis in Smallholder Dairy Cattle and Peridomestic Rodents in Kandy, Sri Lanka

Leptospirosis is an important bacterial zoonotic disease globally and one of the notifiable diseases in Sri Lanka. Other than human leptospirosis, little information is available on leptospirosis in domestic and feral animals in Sri Lanka. Thus, this study attempted to determine the prevalence and carrier status of leptospirosis in small-holder dairy cattle and peridomestic rodents to understand the impact of the disease on public health in Kandy, Sri Lanka. Cattle and rodent samples were collected from the Yatinuwara and Udunuwara divisional secretaries in Kandy. Serum samples were analyzed for the presence of antileptospiral antibodies using microscopic agglutination test. DNA was extracted from cattle urine and rodent kidney tissue samples, in which polymerase chain reaction was carried out to detect the Leptospira flaB gene. The cattle in 19 (38.8%) of the 49 farms harbored antileptospiral antibodies. Out of 113 cattle serum samples, 23 (20.3%) were positive; 17 (73.9%) and 6 (26.1%) reacted with serogroups Sejroe and Hebdomadis, respectively. Out of the 74 rodent samples, 13 (17.5%) were positive; 8 (61.5%) and 4 (30.8%) had reactions to serogroups Javanica and Icterohaemorrhagiae, respectively. Leptospiral DNA was detected in one cattle urine sample and identified as Leptospira interrogans. This study revealed a high prevalence of leptospirosis in cattle and rodents in Kandy. These animals were infected with a wide array of leptospiral serogroups, which are consistent with the research findings observed in humans in Kandy. Overall, serological data indicate that relative to rodents, cattle may be a more significant reservoir for human transmission and a greater source of potential risk to local agricultural communities

Erythematous or dusky red discrete skin rash in both upper and lower limbs involving hands and feet.

<p>Erythematous or dusky red discrete skin rash in both upper and lower limbs involving hands and feet.</p

Cutaneous Manifestations of Spotted Fever Rickettsial Infections in the Central Province of Sri Lanka: A Descriptive Study

<div><p>Background</p><p>Characteristic skin lesions play a key role in clinical diagnosis of spotted fever group rickettsioses and this study describes these cutaneous manifestations along with basic histological features.</p><p>Methods and Findings</p><p>Study was conducted at Medical Unit, Teaching Hospital, Peradeniya, from November 2009 to October 2011, where a prospective data base of all rickettsial infections is maintained. Confirmation of diagnosis was made when IgM and IgG immunofluorescent antibody titre of 1/32 and >1/256 respectively. Of the 210 clinical cases, 134 had cutoff antibody titers for <i>Rickettsia conorii</i> antigen for confirmation. All these 134 patients had fever and skin rash, and of them 132(98%) had discrete maculopapular rash while eight (6%) had fern leaf type skin necrosis. Eight patients (6%) had healed tick bite marks. Average size of a skin lesion was 5 mm and rash involved 52% of body surface, distributed mainly in limbs and back of the chest. Generally the facial and leg skin was slightly oedematous particularly in old aged patients. Sixteen patients (12%) had pain and swelling of ankle joints where swelling extended to feet and leg. Biopsies from skin rash of six patients showed evidence of cutaneous vasculitis and of them, 247 bp region of the 17-kDa spotted fever group specific protein antigen was amplified using PCR.</p><p>Conclusions</p><p>A discrete maculopapular rash and occasional variations such as fern leaf shape necrosis and arthritis are found in spotted fever group. Histology found vasculitis as the pathology of these lesions.</p></div

Fern leaf type skin necrosis in legs.

<p>Fern leaf type skin necrosis in legs.</p

Cutaneous histopathology of maculopapular skin rash, (A) <i>Black arrow-</i> Exocytosis of extravasated lymphocytes.

<p><i>White arrow</i> – Focal swelling of endothelium (haematoxyline-eosin; original magnification ×200), (B) Arrows indicate the vacuolations in the basal cells of epidermis, (C) <i>Black arrow</i> – Ectatic upper dermal blood vessels. <i>White arrow</i> – extravasated red blood cells, (D) <i>Black arrows</i> – Lymphocytic infiltrates in perivascular spaces. <i>White arrows</i> – Focal swelling of endothelium and ectatic upper dermal blood vessels, (E) <i>White arrow</i> – Fibrinoid necrosis of a dermal vessel wall. <i>Black arrow</i> – Presence of fibrin thrombi. (hematoxyline-eosin; original magnification ×800). Scale bars = 50 µm.</p

Tick bite marks found in patients, (A) a tick bite mark, ten days old, (B) a scarred tick bite mark, (C) a recent tick bite mark.

<p>Tick bite marks found in patients, (A) a tick bite mark, ten days old, (B) a scarred tick bite mark, (C) a recent tick bite mark.</p

Details of IFA serology (IgG and IgM) in SFG cases.

a<p>Percentages were calculated, out of the total number of 210 patients.</p>b<p>High titre positive cases presented in detail in this study.</p>c<p>Group with negative seroreactivity.</p><p>Details of IFA serology (IgG and IgM) in SFG cases.</p