Nipah virus (NiV) has become one of the most worrisome zoonotic agents in global health due to its high case fatality, wide host spectrum, capability to induce severe encephalitis as well as respiratory disease, and reportedly, has the potential of person-to-person transmission [1]. Introduced as an outbreak threat in South and Southeast Asia, most recently in 2018, there has been an outbreak of NiV, which was originally described as widespread in the outbreak in 1998-99 in Malaysia and Singapore, and there have been recurrent outbreaks of the pathogen in Bangladesh and India due to spillover by Pteropus fruit bats and occasional secondary transmission between humans [2]. Nipah virus is still a priority pathogen due to its combined severity of outbreaks with the lack of approved specific treatment or a vaccine and remains a plausible future pandemic threat as opposed to being merely another infrequent zoonosis. The Nipah virus (NiV) disease is a viral pathology, which entered Southeast Asia, is caused by negative single-stranded RNA virus with the length of 18,000 nucleotides, which belongs to the family Paramyxoviridae and genus Henipavirus [3-5]. Human beings can also be infected by other species of this genus such as Ghanaian bat virus, Mojiang virus and Hendra virus (HeV) [1]. The viral strain [2] NiV is the cause of the most commonly high morbidity and mortality rate in humans and that is frequently recurring in Bangladesh, India or the Philippines first identified in the 1998-1999 outbreaks in Malaysia and Singapore. The fact that NiV has caused human fatalities up to 70% [3], has made them designated as risk-group 4 pathogens, and necessitates the work on such viruses to be classified under Biosafety Level 4 (BSL-4) facilities. BSL categories take into account the lethality of the disease and availability of prevention and curative treatment in this instance, neither of them can be found with NiV or HeV. NiV and HeV genomes are around 80 per cent of the same with regard to the nucleotides [4] and thus the diagnostics tests do cross-react in the case of the type of RNA sequence being targeted. Similar to other paramyxoviruses, the following proteins are coded by the genome: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein F (F), glycoprotein G (G), and RNA polymerase which is the large protein (L). The non-structural proteins C, V, and W needed in the NiV pathogenicity are encoded by P gene [5]. The viral proteins contained in the replicative complex that includes nucleoprotein, phosphoprotein and the polymerase L and encased by a lipid bi-layered envelope containing the attachment protein G and the fusion F protein are bound with the RNA genome [6]. The NiV receptor is ephrin-B2 which is present in the endothelial cells and neurons [7,8,9]. Alarming about Nipah is not so much its lethality but how it is at the confluence of ecological disruption, exposure of animal to human interface, poor surveillance measures, and late laboratory confirmation. Infection in humans might happen due to direct contact with infected bats or any intermediate host, due to the ingestion of contaminated food items like raw date palm sap, or through a close contact with an infected patient including in a healthcare setting. It is an indicator that the virus already has a number of the ingredients linked with amplification of outbreaks [10-12]. Even though it is not as efficient as the transmission of more conventional respiratory pandemic viruses, the recurrent spillover events and clustered dissemination of NiV continue to place it on the short list of pathogens that need serious preparedness consideration [13]. One of the most significant issues with the Nipah control is the fact that the early clinical manifestation is frequently not specific. Before patients progress to acute encephalitis, respiratory distress, seizures, or coma, they might first experience fever, headache, myalgia, sore throat, cough, vomiting or other general symptoms. It is just the reason why early diagnosis is so sensitive: the initial stage can be similar to numerous other endemic infectious diseases, in particular, in low-resource epidemic conditions [14]. It can be seen that, by the time suspicion is strong enough to lead to confirmatory testing, chances of timely isolation, contact tracing, and focused containment may be already fading away [15]. The present diagnostic approaches are predominantly based on real-time reverse transcription polymerase chain reaction (RT-PCR) in the acute stage and serological, like ELISA, in the later illness or following the recovery [16]. RT-PCR has been regarded as the new standard laboratory practice because the test can identify viral RNA in samples that include throat and nasal swabs, blood, cerebrospinal fluid, and urine whereas antibody-based tests can be used to confirm retrospectively and serosurveillance. The techniques however have limitations in the fact that they require specialized laboratories, biosafety measures, qualified staff, logistics in transportation and also the issue of getting suitable samples at the right time when the disease is at its right stage [17]. Nipah outbreaks also have constraints in most of the settings where they arise, making the issue of diagnosis not a technical issue, but rather a systems failure issue [18-20].

Objective

This review aims to evaluate the current diagnostic strategies for Nipah virus and identify gaps that may affect early detection and outbreak control.

This study was conducted as a narrative review article from 1998 to 2026. A comprehensive literature search was performed using major electronic databases including PubMed, Scopus, Web of Science, Google Scholar, and ScienceDirect. The search focused on identifying relevant peer-reviewed articles, surveillance reports, outbreak investigations, and guidelines related to Nipah virus diagnostics and early detection.

Search Keywords

The search was conducted using combinations of keywords and Boolean operators such as “Nipah virus,” “NiV diagnosis,” “Nipah virus detection,” “RT-PCR for Nipah virus,” “serological diagnosis of Nipah virus,” “early detection of Nipah virus,” and “Nipah virus surveillance.” These keywords were combined using operators such as AND and OR to refine the search results and retrieve the most relevant studies. Studies were included in the review if they met the following criteria: Published in English language Published between 1996 and 2025

• Focused diagnostic methods, surveillance, laboratory detection, or early identification of Nipah virus
• Included original research articles, systematic reviews, outbreak reports, and guidelines from recognized public health

organizations such as the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC).

There were two stages to the selection of relevant articles. The first step consisted of screening the titles and abstracts of the identified articles to determine their relevance to the subject. In the second phase, the complete texts of selected studies were carefully examined to determine whether or not they should be included in the analysis. Relevant information from the selected studies was extracted systematically.  The types of clinical samples that were analyzed, the sensitivity and specificity of diagnostic assays, the requirements for laboratory infrastructure, and the limitations that are associated with early detection during outbreaks were some of the most important data. The extracted data were then broken down into themes like molecular diagnostic techniques, serological testing, new rapid diagnostic technologies, surveillance systems, and diagnostic difficulties. Qualitatively synthesising the collected data, a comprehensive overview of the Nipah virus's current diagnostic methods and significant gaps in early detection systems were discovered. This thematic analysis shed light on the advantages and disadvantages of currently available diagnostic tools and revealed areas in which additional research and technological advancement are required for effective outbreak preparedness.

Table 1: Chronology of Major Nipah Virus Outbreaks (1998–2026)

Although Pakistan has not experienced a confirmed Nipah virus outbreak, the presence of fruit bat reservoirs, dense human populations, livestock interactions, and regional proximity to endemic countries (India and Bangladesh) suggests that the country remains potentially vulnerable to future spillover events, making early detection systems and diagnostic preparedness critically important.

Current Diagnostic Modalities Identified in the Literature

Across the literature, the most frequently reported diagnostic methods were real-time reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) for IgM and IgG antibodies, virus isolation, neutralization assays, immunohistochemistry, and sequencing-based methods. Among these, RT-PCR was consistently described as the preferred method for early confirmation of acute infection because it directly detects viral RNA in respiratory specimens, blood, cerebrospinal fluid, urine, and sometimes tissue samples. ELISA was repeatedly reported as useful for later diagnosis, retrospective case confirmation, and serosurveillance rather than the earliest detection window.

Table 2. Summary of Diagnostic Approaches Reported in the Literature for Nipah Virus Detection

Table 3. Clinical Specimens Used for Nipah Virus Diagnosis in Reviewed Studies

This table summarizes major outbreak studies and diagnostic strategies used globally between 1998 and 2026, highlighting the consistent reliance on molecular detection methods (RT-PCR), supportive serological assays, and genomic sequencing in recent investigations. The literature shows that early laboratory confirmation combined with rapid surveillance and contact tracing remains the most effective strategy for controlling Nipah virus outbreaks, emphasizing the importance of strengthening diagnostic infrastructure in high-risk regions.

Due to its high case fatality rate, recurrent outbreaks, and potential for human-to-human transmission, the nipah virus remains one of the emerging zoonotic pathogens that raise the most concerns. Key obstacles to early detection were identified through this systematic review of the diagnostic strategy literature. The results show that, despite significant advancements in laboratory diagnostics, significant gaps remain in the prompt identification and containment of outbreaks. The review emphasizes that Nipah virus detection continues to be based on molecular diagnostic methods, particularly real-time reverse transcription polymerase chain reaction (RT-PCR). During the acute phase of infection, RT-PCR permits the direct identification of viral RNA in clinical specimens like throat swabs, blood, cerebrospinal fluid, and urine. Rapid RT-PCR confirmation was consistently found to facilitate prompt public health interventions like patient isolation and contact tracing in several outbreak investigations [21]. However, despite its high sensitivity and specificity, RT-PCR's widespread application is constrained by the requirement of specialized labs, trained personnel, and stringent biosafety protocols. In the literature, serological testing techniques like the enzyme-linked immunosorbent assay (ELISA) for IgM and IgG antibodies were also frequently mentioned. In the later stages of illness or for retrospective epidemiological studies, these assays are especially useful for confirming infection. However, because antibodies may not be detectable during the initial phase of infection, serology alone is insufficient for early diagnosis [22]. Consequently, for accurate Nipah virus infection diagnosis and surveillance, a combination of molecular and serological methods is frequently recommended. The strong ecological association between Nipah virus outbreaks and the presence of fruit bats of the genus Pteropus, which serve as the natural reservoir for the virus, is another important finding from the review [23]. Transmission to humans may occur through direct contact with infected animals, consumption of bat-contaminated food products such as raw date palm sap, or close contact with infected individuals.  There was evidence of human-to-human transmission in several outbreaks, particularly those that were reported in Bangladesh and India [24]. This pattern of transmission raises concerns about the virus’s potential to cause larger epidemics if early detection and containment measures are not implemented promptly.  The literature consistently identifies several barriers to early detection [25-27] in spite of advancements in diagnostic technologies. The nonspecific nature of the initial clinical symptoms of Nipah virus infection, which frequently resemble other febrile illnesses like influenza or viral encephalitis, is one of the major obstacles. In particular in regions where the disease is not frequently encountered, this similarity can delay clinical suspicion and diagnostic testing. Additionally, a lack of laboratory infrastructure in many outbreak-prone regions can delay the confirmation of cases and impede prompt public health responses. In addition, recent studies have emphasized the significance of developing rapid point-of-care diagnostic tools and strengthening surveillance systems [28]. Multiplex PCR assays, genomic sequencing, and loop-mediated isothermal amplification (LAMP) are promising new technologies for enhancing outbreak monitoring and early detection. Particularly in low-resource settings where traditional laboratory infrastructure may be limited, these technologies have the potential to provide faster and more accessible diagnostic options. Another important implication of the findings is the need for improved integration between ecological surveillance, clinical detection, and laboratory diagnostics [29].  Spillover events can be detected earlier if bat populations are monitored, high-risk human–animal interactions are identified, and zoonotic disease surveillance is strengthened. It is becoming increasingly clear that integrated "One Health" strategies are necessary for the prevention of new infectious diseases, such as the Nipah virus. There are some restrictions on this systematic review. The inclusion of only English-language studies may have excluded relevant research that was available in other languages [30-32]. Additionally, the majority of the included studies were narrative reviews, outbreak investigations, or observational reports rather than large-scale diagnostic accuracy studies, limiting the strength and comparability of the evidence. Due to the relatively low number of confirmed Nipah virus outbreaks worldwide, many studies used small sample sizes, which limited the findings' generalizability. Furthermore, studies with significant results are more likely to be published than those with negative or inconclusive results, which may lead to publication bias. Case detection and reporting may also be affected by differences in countries' surveillance systems, diagnostic capabilities, and healthcare infrastructure, which could impact the overall evaluation of the diagnostic strategies and early detection gaps identified in this review.

In conclusion, Nipah virus remains a significant emerging zoonotic pathogen with a high mortality rate and the potential to cause severe outbreaks. This review highlights that molecular diagnostic techniques, particularly RT-PCR, currently represent the most reliable method for early detection, while serological assays provide important support for confirmation and epidemiological investigations. However, several gaps remain in the early diagnosis of the disease, including nonspecific clinical presentation, limited laboratory infrastructure in outbreak-prone regions, and the lack of widely available rapid point-of-care diagnostic tools. Strengthening surveillance systems, expanding diagnostic capacity, and developing accessible rapid diagnostic technologies are essential steps for improving early detection and outbreak control

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